Oligonucleotides associated with antibodies

ABSTRACT

Disclosed herein include systems, methods, compositions, and kits for determining protein expression and gene expression simultaneously and for sample indexing. In some embodiments, an oligonucleotide associated with a cellular component-binding reagent (e.g., an antibody) comprises one or more of a unique molecular label sequence, a primer adapter, antibody-specific barcode sequence, an alignment sequence, and/or a poly(A) sequence. In some embodiments, the oligonucleotide is associated with the cellular component-binding reagent via a linker (e.g., 5AmMC12).

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application No. 62/796,018, filed Jan. 23, 2019, the contentof this related application is incorporated herein by reference in itsentirety for all purposes.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledSequence_Listing_68EB_298700_US, created on Jan. 21, 2020, which is 4kilobytes in size. The information in the electronic format of theSequence Listing is incorporated herein by reference in its entirety.

BACKGROUND Field

The present disclosure relates generally to the field of molecularbiology, for example identifying cells of different samples anddetermining protein expression profiles in cells using molecularbarcoding.

Description of the Related Art

Current technology allows measurement of gene expression of single cellsin a massively parallel manner (e.g., >10000 cells) by attaching cellspecific oligonucleotide barcodes to poly(A) mRNA molecules fromindividual cells as each of the cells is co-localized with a barcodedreagent bead in a compartment. Gene expression may affect proteinexpression. Protein-protein interaction may affect gene expression andprotein expression. There is a need for systems and methods that canquantitatively analyze protein expression in cells, and simultaneouslymeasure protein expression and gene expression in cells.

SUMMARY

Some embodiments disclosed herein provide methods for sampleidentification, comprising: contacting each of a plurality of sampleswith a sample indexing composition of a plurality of sample indexingcompositions, respectively, wherein each of the plurality of samplescomprises one or more cells each comprising one or more cellularcomponent targets, wherein the sample indexing composition comprises acellular component-binding reagent associated with a sample indexingoligonucleotide, wherein the cellular component-binding reagent iscapable of specifically binding to at least one of the one or morecellular component targets, wherein the sample indexing oligonucleotidecomprises a sample indexing sequence, and wherein sample indexingsequences of at least two sample indexing compositions of the pluralityof sample indexing compositions comprise different sequences; andidentifying sample origin of at least one cell of the one or more cellsbased on the sample indexing sequence of at least one sample indexingoligonucleotide of the plurality of sample indexing compositions.

In some embodiments, the sample indexing oligonucleotide comprises afirst molecular label sequence. In some embodiments, the first molecularlabel sequence is 2-20 nucleotides in length. In some embodiments, thefirst molecular label sequences of at least two sample indexingoligonucleotides are different, and wherein the sample indexingsequences of the at least two sample indexing oligonucleotides areidentical. In some embodiments, the first molecular label sequences ofat least two sample indexing oligonucleotides are different, and whereinthe sample indexing sequences of the at least two sample indexingoligonucleotides are different.

In some embodiments, the sample indexing oligonucleotide comprises thesequence of a first universal primer, a complementary sequence thereof,a partial sequence thereof, or a combination thereof. In someembodiments, the first universal primer is 5-50 nucleotides in length.In some embodiments, the first universal primer comprises anamplification primer, a sequencing primer, or a combination thereof. Insome embodiments, the sequencing primer comprises a P7 sequencingprimer.

In some embodiments, identifying the sample origin of the at least onecell comprises: providing a plurality of barcodes. In some embodiments,identifying the sample origin of the at least one cell comprises:barcoding sample indexing oligonucleotides of the plurality of sampleindexing compositions using a plurality of barcodes to generate aplurality of barcoded sample indexing oligonucleotides; obtainingsequencing data of the plurality of barcoded sample indexingoligonucleotides; and identifying the sample origin of the cell based onthe sample indexing sequence of at least one barcoded sample indexingoligonucleotide of the plurality of barcoded sample indexingoligonucleotides in the sequencing data. In some embodiments,identifying the sample origin of the at least one cell comprisesidentifying the presence or absence of the sample indexing sequence ofat least one sample indexing oligonucleotide, or of at least onebarcoded sample indexing oligonucleotide, in the sequencing data. Insome embodiments, identifying the presence or absence of the sampleindexing sequence comprises: replicating the at least one sampleindexing oligonucleotide, the at least one barcoded sample indexingoligonucleotide, or a product thereof, using the first universal primer,a first primer comprising the sequence of the first universal primer, ora combination thereof, to generate a plurality of replicated sampleindexing oligonucleotides; obtaining sequencing data of the plurality ofreplicated sample indexing oligonucleotides; and identifying the sampleorigin of the cell based on the sample indexing sequence of a replicatedsample indexing oligonucleotide of the plurality of replicated sampleindexing oligonucleotides that correspond to the at least one sampleindexing oligonucleotide, or the least one barcoded sample indexingoligonucleotide, in the sequencing data.

In some embodiments, replicating the at least one sample indexingoligonucleotide to generate the plurality of replicated sample indexingoligonucleotides comprises: prior to replicating the at least onebarcoded sample indexing oligonucleotide, ligating a replicating adaptorto the at least one barcoded sample indexing oligonucleotide, andwherein replicating the at least one barcoded sample indexingoligonucleotide comprises replicating the at least one barcoded sampleindexing oligonucleotide using the replicating adaptor ligated to the atleast one barcoded sample indexing oligonucleotide to generate theplurality of replicated sample indexing oligonucleotides. In someembodiments, replicating the at least one sample indexingoligonucleotide to generate the plurality of replicated sample indexingoligonucleotides comprises: prior to replicating the at least onebarcoded sample indexing oligonucleotide, contacting a barcode of theplurality of barcodes with the at least one sample indexingoligonucleotide, or a product of the sample indexing oligonucleotide, togenerate the barcode hybridized to the sample indexing oligonucleotide,or a product thereof; and extending the barcode hybridized to the sampleindexing oligonucleotide, or the product of the sample indexingoligonucleotide, to generate the barcoded sample indexingoligonucleotide of the plurality of barcoded sample indexingoligonucleotides, and wherein replicating the at least one sampleindexing oligonucleotide comprises replicating the barcoded sampleindexing oligonucleotide, or a product thereof, using the firstuniversal primer, a first primer comprising the sequence of the firstuniversal primer, the second universal primer, a second primercomprising the sequence of the second universal primer, or a combinationthereof, to generate the plurality of replicated sample indexingoligonucleotides.

In some embodiments, the product of the sample indexing oligonucleotidecomprises a complementary sequence of the at least one sample indexingoligonucleotide, or a subsequence thereof, and the sequence of thebarcode, or a subsequence thereof. In some embodiments, the sampleindexing oligonucleotide comprises an alignment sequence adjacent to thepoly(dA) region.

In some embodiments, the alignment sequence is one or more nucleotidesin length. In some embodiments, the alignment sequence is two or morenucleotides in length. In some embodiments, the alignment sequencecomprises a guanine, a cytosine, a thymine, a uracil, or a combinationthereof. In some embodiments, the alignment sequence comprises apoly(dT) sequence, a poly(dG) sequence, a poly(dC) sequence, a poly(dU)sequence, or a combination thereof. In some embodiments, the alignmentsequence is 5′ to the poly(dA) region.

In some embodiments, the sample indexing oligonucleotide is associatedwith the cellular component-binding reagent through a linker. In someembodiments, the linker comprises a carbon chain. In some embodiments,the carbon chain comprises 2-30 carbons. In some embodiments, the carbonchain comprises 12 carbons. In some embodiments, the linker comprises 5′amino modifier C12 (5AmMC12), or a derivative thereof. In someembodiments, the cellular component target comprises a protein target.

In some embodiments, the sample indexing sequence is 6-60 nucleotides inlength. In some embodiments, the sample indexing oligonucleotide is50-500 nucleotides in length. In some embodiments, sample indexingsequences of at least 10, 100, or 1000 sample indexing compositions ofthe plurality of sample indexing compositions comprise differentsequences. In some embodiments, the sample indexing oligonucleotide isattached to the cellular component-binding reagent. In some embodiments,the sample indexing oligonucleotide is covalently attached to thecellular component-binding reagent. In some embodiments, the sampleindexing oligonucleotide is conjugated to the cellular component-bindingreagent. In some embodiments, the sample indexing oligonucleotide isconjugated to the cellular component-binding reagent through a chemicalgroup selected from the group consisting of a UV photocleavable group, astreptavidin, a biotin, an amine, and a combination thereof. In someembodiments, the sample indexing oligonucleotide is non-covalentlyattached to the cellular component-binding reagent. In some embodiments,the at least one of the one or more cellular component targets is on acell surface. In some embodiments, the methods comprise removing unboundsample indexing compositions of the plurality of sample indexingcompositions. In some embodiments, removing the unbound sample indexingcompositions comprises washing the one or more cells from each of theplurality of samples with a washing buffer. In some embodiments,removing the unbound sample indexing compositions comprises selectingcells bound to at least one cellular component-binding reagent usingflow cytometry. In some embodiments, the methods comprise comprisinglysing the one or more cells from each of the plurality of samples. Insome embodiments, the sample indexing oligonucleotide is configured tobe detachable from the cellular component-binding reagent. In someembodiments, the methods comprise dissociating the sample indexingoligonucleotide from the cellular component-binding reagent. In someembodiments, dissociating the sample indexing oligonucleotide comprisesdetaching the sample indexing oligonucleotide from the cellularcomponent-binding reagent by UV photocleaving, chemical treatment,heating, enzyme treatment, or any combination thereof. In someembodiments, the dissociating occurs after barcoding the sample indexingoligonucleotides. In some embodiments, the dissociating occurs beforebarcoding the sample indexing oligonucleotides. In some embodiments, thesample indexing oligonucleotide is configured to be non-detachable fromthe cellular component-binding reagent.

In some embodiments, the sample indexing oligonucleotide is nothomologous to genomic sequences of any of the one or more cells, ishomologous to genomic sequences of a species, or a combination thereof.In some embodiments, the species is a non-mammalian species. In someembodiments, a sample of the plurality of samples comprises a pluralityof cells, a plurality of single cells, a tissue, a tumor sample, or anycombination thereof. In some embodiments, the plurality of samplescomprises a mammalian cell, a bacterial cell, a viral cell, a yeastcell, a fungal cell, or any combination thereof.

In some embodiments, the sample indexing oligonucleotide comprises asequence complementary to a capture sequence of a barcode configured tocapture the sequence of the sample indexing oligonucleotide. In someembodiments, the barcode comprises a target-binding region whichcomprises the capture sequence. In some embodiments, the target-bindingregion comprises a poly(dT) region. In some embodiments, the sequence ofthe sample indexing oligonucleotide complementary to the capturesequence comprises a poly(dA) region.

In some embodiments, the cellular component target comprises acarbohydrate, a lipid, a protein, an extracellular protein, acell-surface protein, a cell marker, a B-cell receptor, a T-cellreceptor, a major histocompatibility complex, a tumor antigen, areceptor, an intracellular protein, or any combination thereof. In someembodiments, the cellular component target is selected from a groupcomprising 10-100 different cellular component targets. In someembodiments, the cellular component-binding reagent is associated withtwo or more sample indexing oligonucleotides with an identical sequence.In some embodiments, the cellular component-binding reagent isassociated with two or more sample indexing oligonucleotides withdifferent sample indexing sequences. In some embodiments, the sampleindexing composition comprises a second cellular component-bindingreagent capable of specifically binding to at least one of the one ormore cellular component targets. In some embodiments, the cellularcomponent-binding reagent and the second cellular component-bindingreagent are capable of binding to the same cellular component-bindingtarget of the one or more cellular component targets. In someembodiments, the second cellular component-binding reagent is associatedwith a second sample indexing oligonucleotide comprising a second sampleindexing sequence. In some embodiments, the sample indexing sequence andthe second sample indexing sequence are identical. In some embodiments,the sample indexing sequence and the second sample indexing sequence aredifferent. In some embodiments, the cellular component-binding reagentand the second cellular component-binding reagent are at least 60%, 70%,80%, 90%, or 95% identical in sequence. In some embodiments, thecellular component-binding reagent and the second cellularcomponent-binding reagent are identical. In some embodiments, thecellular component-binding reagent and the second cellularcomponent-binding reagent are different. In some embodiments, thecellular component-binding reagent and the second cellularcomponent-binding reagent are capable of binding to different regions ofthe same cellular component target. In some embodiments, the cellularcomponent-binding reagent and the second cellular component-bindingreagent are capable of binding to different protein targets of the oneor more protein targets, or different cellular component targets of theone or more cellular component targets.

In some embodiments, the methods comprise prior to barcoding the sampleindexing oligonucleotides, pooling the plurality of samples contactedwith the plurality of sample indexing compositions. In some embodiments,a barcode of the plurality of barcodes comprises a target-binding regionand a second molecular label sequence, and wherein the second molecularlabel sequences of at least two barcodes of the plurality of barcodescomprise different second molecule label sequences. In some embodiments,the barcode comprises a cell label sequence, the sequence of a seconduniversal primer, a complementary sequence thereof, a subsequencethereof, or any combination thereof. In some embodiments, thetarget-binding region comprises a poly(dT) region. In some embodiments,the plurality of barcodes is associated with a particle. In someembodiments, at least one barcode of the plurality of barcodes isimmobilized on the particle, partially immobilized on the particle,enclosed in the particle, partially enclosed in the particle, or acombination thereof. In some embodiments, the particle is disruptable.In some embodiments, the particle comprises a bead. In some embodiments,the particle comprises a sepharose bead, a streptavidin bead, an agarosebead, a magnetic bead, a conjugated bead, a protein A conjugated bead, aprotein G conjugated bead, a protein AIG conjugated bead, a protein Lconjugated bead, an oligo(dT) conjugated bead, a silica bead, asilica-like bead, a hydrogel bead, an anti-biotin microbead, ananti-fluorochrome microbead, or any combination thereof, or wherein theparticle comprises a material selected from the group consisting ofpolydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose,gelatin, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene,acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone,and any combination thereof. In some embodiments, the particle comprisesa disruptable hydrogel bead. In some embodiments, the barcodes of theparticle comprise second molecular label sequences selected from atleast 1000, 10000, or a combination thereof, different second molecularlabel sequences. In some embodiments, the second molecular labelsequences of the barcodes comprise random sequences. In someembodiments, the particle comprises at least 10000 barcodes.

In some embodiments, barcoding the sample indexing oligonucleotidesusing the plurality of barcodes comprises: contacting the plurality ofbarcodes with the sample indexing oligonucleotides to generate barcodeshybridized to the sample indexing oligonucleotides; and extending thebarcodes hybridized to the sample indexing oligonucleotides to generatethe plurality of barcoded sample indexing oligonucleotides. In someembodiments, the methods comprise, prior to extending the barcodeshybridized to the sample indexing oligonucleotides, pooling the barcodeshybridized to the sample indexing oligonucleotides, and whereinextending the barcodes hybridized to the sample indexingoligonucleotides comprises extending the pooled barcodes hybridized tothe sample indexing oligonucleotides to generated a plurality of pooledbarcoded sample indexing oligonucleotides. In some embodiments,extending the barcodes comprises extending the barcodes using a DNApolymerase to generate the plurality of barcoded sample indexingoligonucleotides. In some embodiments, extending the barcodes comprisesextending the barcodes using a reverse transcriptase to generate theplurality of barcoded sample indexing oligonucleotides. In someembodiments, the methods comprise amplifying the plurality of barcodedsample indexing oligonucleotides to produce a plurality of amplicons. Insome embodiments, amplifying the plurality of barcoded sample indexingoligonucleotides comprises amplifying, using polymerase chain reaction(PCR), at least a portion of the second molecular label sequence and atleast a portion of the sample indexing oligonucleotide. In someembodiments, obtaining the sequencing data of the plurality of barcodedsample indexing oligonucleotides comprises obtaining sequencing data ofthe plurality of amplicons. In some embodiments, obtaining thesequencing data comprises sequencing at least a portion of the secondmolecular label sequence and at least a portion of the sample indexingoligonucleotide.

In some embodiments, barcoding the sample indexing oligonucleotidesusing the plurality of barcodes to generate the plurality of barcodedsample indexing oligonucleotides comprises stochastically barcoding thesample indexing oligonucleotides using a plurality of stochasticbarcodes to generate a plurality of stochastically barcoded sampleindexing oligonucleotides. In some embodiments, the methods comprise:barcoding a plurality of targets of the cell using the plurality ofbarcodes to generate a plurality of barcoded targets, wherein each ofthe plurality of barcodes comprises a cell label sequence, and whereinat least two barcodes of the plurality of barcodes comprise an identicalcell label sequence; and obtaining sequencing data of the barcodedtargets. In some embodiments, barcoding the plurality of targets usingthe plurality of barcodes to generate the plurality of barcoded targetscomprises: contacting copies of the targets with target-binding regionsof the barcodes; and reverse transcribing the plurality targets usingthe plurality of barcodes to generate a plurality of reverse transcribedtargets. In some embodiments, the methods comprise: prior to obtainingthe sequencing data of the plurality of barcoded targets, amplifying thebarcoded targets to generate a plurality of amplified barcoded targets.In some embodiments, amplifying the barcoded targets to generate theplurality of amplified barcoded targets comprises: amplifying thebarcoded targets by polymerase chain reaction (PCR). In someembodiments, barcoding the plurality of targets of the cell using theplurality of barcodes to generate the plurality of barcoded targetscomprises stochastically barcoding the plurality of targets of the cellusing a plurality of stochastic barcodes to generate a plurality ofstochastically barcoded targets.

Some embodiments disclosed herein provide a plurality of sample indexingcompositions, wherein each of the plurality of sample indexingcompositions comprises a cellular component-binding reagent associatedwith a sample indexing oligonucleotide, wherein the cellularcomponent-binding reagent is capable of specifically binding to at leastone cellular component target, wherein the sample indexingoligonucleotide comprises a sample indexing sequence for identifyingsample origin of one or more cells of a sample, and wherein sampleindexing sequences of at least two sample indexing compositions of theplurality of sample indexing compositions comprise different sequences.

In some embodiments, the sample indexing oligonucleotide comprises afirst molecular label sequence. In some embodiments, the first molecularlabel sequence is 2-20 nucleotides in length. In some embodiments, thefirst molecular label sequences of at least two sample indexingoligonucleotides are different, and wherein the sample indexingsequences of the at least two sample indexing oligonucleotides areidentical. In some embodiments, the first molecular label sequences ofat least two sample indexing oligonucleotides are different, and whereinthe sample indexing sequences of the at least two sample indexingoligonucleotides are different. In some embodiments, the sample indexingoligonucleotide comprises the sequence of a first universal primer, acomplementary sequence thereof, a partial sequence thereof, or acombination thereof. In some embodiments, the first universal primer is5-50 nucleotides in length. In some embodiments, the first universalprimer comprises an amplification primer, a sequencing primer, or acombination thereof. In some embodiments, the sequencing primercomprises a P7 sequencing primer. In some embodiments, the sampleindexing oligonucleotide comprises an alignment sequence adjacent to thepoly(dA) region. In some embodiments, the alignment sequence is one ormore nucleotides in length. In some embodiments, the alignment sequenceis two or more nucleotides in length. In some embodiments, the alignmentsequence comprises a guanine, a cytosine, a thymine, a uracil, or acombination thereof. In some embodiments, the alignment sequencecomprises a poly(dT) sequence, a poly(dG) sequence, a poly(dC) sequence,a poly(dU) sequence, or a combination thereof. In some embodiments, thealignment sequence is 5′ to the poly(dA) region. In some embodiments,the sample indexing oligonucleotide is associated with the cellularcomponent-binding reagent through a linker. In some embodiments, thelinker comprises a carbon chain. In some embodiments, the carbon chaincomprises 2-30 carbons. In some embodiments, the carbon chain comprises12 carbon. In some embodiments, the linker comprises 5′ amino modifierC12 (5AmMC12), or a derivative thereof. In some embodiments, thecellular component target comprises a protein target.

In some embodiments, the sample indexing sequence is 6-60 nucleotides inlength. In some embodiments, the sample indexing oligonucleotide is50-500 nucleotides in length. In some embodiments, the sample indexingsequences of at least 10, 100, or 1000 sample indexing compositions ofthe plurality of sample indexing compositions comprise differentsequences. In some embodiments, the sample indexing oligonucleotide isattached to the cellular component-binding reagent. In some embodiments,the sample indexing oligonucleotide is covalently attached to thecellular component-binding reagent. In some embodiments, the sampleindexing oligonucleotide is conjugated to the cellular component-bindingreagent. In some embodiments, the sample indexing oligonucleotide isconjugated to the cellular component-binding reagent through a chemicalgroup selected from the group consisting of a UV photocleavable group, astreptavidin, a biotin, an amine, and a combination thereof. In someembodiments, the sample indexing oligonucleotide is non-covalentlyattached the cellular component-binding reagent.

In some embodiments, the sample indexing composition comprises a secondcellular component-binding reagent capable of specifically binding to atleast one of the one or more cellular component targets. In someembodiments, the cellular component-binding reagent and the secondcellular component-binding reagent are capable of binding to the samecellular component-binding target of the one or more cellular componenttargets. In some embodiments, the second cellular component-bindingreagent is associated with a second sample indexing oligonucleotidecomprising a second sample indexing sequence. In some embodiments, thesample indexing sequence and the second sample indexing sequence areidentical. In some embodiments, the sample indexing sequence and thesecond sample indexing sequence are different. In some embodiments, thecellular component-binding reagent and the second cellularcomponent-binding reagent are at least 60%, 70%, 80%, 90%, or 95%identical in sequence. In some embodiments, the cellularcomponent-binding reagent and the second cellular component-bindingreagent are identical. In some embodiments, the cellularcomponent-binding reagent and the second cellular component-bindingreagent are different. In some embodiments, the cellularcomponent-binding reagent and the second cellular component-bindingreagent are capable of binding to different regions of the same cellularcomponent target. In some embodiments, the cellular component-bindingreagent and the second cellular component-binding reagent are capable ofbinding to different cellular component targets of the one or morecellular component targets.

In some embodiments, the sample indexing oligonucleotide is nothomologous to genomic sequences of any of the one or more cells. In someembodiments, at least one sample of the plurality of samples comprisesone or more single cells, a plurality of cells, a tissue, a tumorsample, or any combination thereof. In some embodiments, the samplecomprises a mammalian sample, a bacterial sample, a viral sample, ayeast sample, a fungal sample, or any combination thereof. In someembodiments, the cellular component target comprises a cell-surfaceprotein, a cell marker, a B-cell receptor, a T-cell receptor, a majorhistocompatibility complex, a tumor antigen, a receptor, or anycombination thereof. In some embodiments, the cellular component targetis selected from a group comprising 10-100 different cellular componenttargets.

Some embodiments disclosed herein provide methods for measuring cellularcomponent expression in cells, comprising: contacting a plurality ofcellular component-binding reagents with a plurality of cells comprisinga plurality of cellular component targets, wherein each of the pluralityof cellular component-binding reagents comprises a cellularcomponent-binding reagent specific oligonucleotide comprising a uniqueidentifier sequence for the cellular component-binding reagent, andwherein the cellular component-binding reagent is capable ofspecifically binding to at least one of the plurality of cellularcomponent targets; extending barcodes hybridized to the cellularcomponent-binding reagent specific oligonucleotides, or productsthereof, to produce a plurality of labeled nucleic acids, wherein eachof the labeled nucleic acid comprises a unique identifier sequence, or acomplementary sequence thereof, and a first molecular label sequence, ora complementary sequence thereof; and obtaining sequence information ofthe plurality of labeled nucleic acids, a complementary sequencethereof, or a portion thereof to determine the number of copies of atleast one cellular component target of the plurality of cellularcomponent targets in one or more of the plurality of cells.

In some embodiments, the methods comprise prior to extending barcodes:partitioning the plurality of cells associated with the plurality ofcellular component-binding reagents to a plurality of partitions,wherein a partition of the plurality of partitions comprises a singlecell from the plurality of cells associated with the cellularcomponent-binding reagents; in the partition comprising the single cell,contacting a barcoding particle with the cellular component specificoligonucleotides, wherein the barcoding particle comprises a pluralityof barcodes each comprising a target binding region and a firstmolecular label sequence, and wherein two barcodes of the plurality ofbarcodes comprise different first molecular label sequences. In someembodiments, the number of unique first molecular label sequencesassociated with the unique identifier sequence for the cellularcomponent-binding reagent capable of specifically binding to the atleast one cellular component target in the sequencing data indicates thenumber of copies of the at least one cellular component target in theone or more of the plurality of cells.

In some embodiments, the cellular component-binding reagent specificoligonucleotide comprises a second molecular label sequence. In someembodiments, the second molecular label sequence is 2-20 nucleotides inlength. In some embodiments, the second molecular label sequences of atleast two cellular component-binding reagent specific oligonucleotidesare different, and wherein the unique identifier sequences of the atleast two cellular component-binding reagent specific oligonucleotidesare identical. In some embodiments, the second molecular label sequencesof at least two cellular component-binding reagent specificoligonucleotides are different, and wherein the unique identifiersequences of the at least two cellular component-binding reagentspecific oligonucleotides are different. In some embodiments, the numberof unique second molecular label sequences associated with the uniqueidentifier sequence for the cellular component-binding reagent capableof specifically binding to the at least one cellular component target inthe sequencing data indicates the number of copies of the at least onecellular component target in the one or more of the plurality of cells.

In some embodiments, the cellular component-binding reagent specificoligonucleotide comprises the sequence of a first universal primer, acomplementary sequence thereof, a partial sequence thereof, or acombination thereof. In some embodiments, the first universal primer is5-50 nucleotides in length. In some embodiments, the first universalprimer comprises an amplification primer, a sequencing primer, or acombination thereof. In some embodiments, the sequencing primercomprises a P7 sequencing primer. In some embodiments, obtainingsequencing information of the plurality of labeled nucleic acids or aportion thereof comprises subjecting the labeled nucleic acids to one ormore reactions (e.g., a PCR amplification, reaction, a reversetranscription reaction, a hybridization and extension reaction, aligation reaction, or any combination thereof) to generate a set ofnucleic acids for nucleic acid sequencing. In some embodiments, each ofthe barcodes comprises a cell label, a second universal primer, anamplification adaptor, a sequencing adaptor, or a combination thereof.In some embodiments, subjecting the labeled nucleic acids to the one ormore reactions comprises subjecting the labeled nucleic acids to anamplification reaction, using the first universal primer, a first primercomprising the sequence of the first universal primer, the seconduniversal primer, a second primer comprising the sequence of the seconduniversal primer, or a combination thereof, to generate the set ofnucleic acids for nucleic acid sequencing.

In some embodiments, the cellular component-binding reagent specificoligonucleotide comprises an alignment sequence adjacent to the poly(dA)region. In some embodiments, the alignment sequence is one or morenucleotides in length. In some embodiments, the alignment sequence istwo or more nucleotides in length. In some embodiments, the alignmentsequence comprises a guanine, a cytosine, a thymine, a uracil, or acombination thereof. In some embodiments, the alignment sequencecomprises a poly(dT) sequence, a poly(dG) sequence, a poly(dC) sequence,a poly(dU) sequence, or a combination thereof. In some embodiments, thealignment sequence is 5′ to the poly(dA) region. In some embodiments,the cellular component-binding reagent specific oligonucleotide isassociated with the cellular component-binding reagent through a linker.In some embodiments, the linker comprises a carbon chain. In someembodiments, the carbon chain comprises 2-30 carbons. In someembodiments, the carbon chain comprises 12 carbons. In some embodiments,the linker comprises 5′ amino modifier C12 (5AmMC12), or a derivativethereof.

In some embodiments, the plurality of cellular component targetscomprises a plurality of protein targets, and wherein the cellularcomponent-binding reagent is capable of specifically binding to at leastone of the plurality of protein targets. In some embodiments, thecellular component-binding reagent specific oligonucleotide isassociated with the cellular component-binding reagent. In someembodiments, the cellular component-binding reagent specificoligonucleotide is covalently attached to the cellular component-bindingreagent. In some embodiments, the cellular component-binding reagentspecific oligonucleotide is conjugated to the cellular component-bindingreagent. In some embodiments, the cellular component-binding reagentspecific oligonucleotide is conjugated to the cellular component-bindingreagent through a chemical group selected from the group consisting of aUV photocleavable group, a streptavidin, a biotin, an amine, and acombination thereof. In some embodiments, the cellular component-bindingreagent specific oligonucleotide is non-covalently attached to thecellular component-binding reagent. In some embodiments, the cellularcomponent-binding reagent specific oligonucleotide is configured to bedetachable from the cellular component-binding reagent. In someembodiments, the methods comprise dissociating the cellularcomponent-binding reagent specific oligonucleotide from the cellularcomponent-binding reagent. In some embodiments, dissociating thecellular component-binding reagent specific oligonucleotide comprisesdetaching the cellular component-binding reagent specificoligonucleotide from the cellular component-binding reagent by UVphotocleaving, chemical treatment, heating, enzyme treatment, or anycombination thereof. In some embodiments, the dissociating occurs afterbarcoding the cellular component-binding reagent specificoligonucleotide. In some embodiments, the dissociating occurs beforebarcoding the cellular component-binding reagent specificoligonucleotide. In some embodiments, the cellular component-bindingreagent specific oligonucleotide is configured to be non-detachable fromthe cellular component-binding reagent.

In some embodiments, the plurality of cellular component-bindingreagents comprises a second cellular component-binding reagent. In someembodiments, the cellular component-binding reagent and the secondcellular component-binding reagent are at least 60%, 70%, 80%, 90%, or95% identical in sequence. In some embodiments, the cellularcomponent-binding reagent and the second cellular component-bindingreagent are identical. In some embodiments, the cellularcomponent-binding reagent and the second cellular component-bindingreagent are different. In some embodiments, the cellular componenttargets of the cellular component-binding reagent and the secondcellular component-binding reagent are identical. In some embodiments,the cellular component-binding reagent and the second cellularcomponent-binding reagent are capable of binding to different regions ofa cellular component target. In some embodiments, the cellular componenttargets of the cellular component-binding reagent and the secondcellular component-binding reagent are different.

In some embodiments, the cellular component-binding reagent specificoligonucleotide comprises a sequence complementary to a capture sequenceof a barcode configured to capture the sequence of the cellularcomponent-binding reagent specific oligonucleotide. In some embodiments,the barcode comprises a target-binding region which comprises thecapture sequence. In some embodiments, the target-binding regioncomprises a poly(dT) region. In some embodiments, the sequence of thecellular component-binding reagent specific oligonucleotidecomplementary to the capture sequence comprises a poly(dA) region.

In some embodiments, the methods comprise after contacting the pluralityof cellular component-binding reagents with the plurality of cells,removing one or more cellular component-binding reagents of theplurality of cellular component-binding reagents that are not contactedwith the plurality of cells. In some embodiments, removing the one ormore cellular component-binding reagents not contacted with theplurality of cells comprises: removing the one or more cellularcomponent-binding reagents not contacted with the respective at leastone of the plurality of cellular component targets. In some embodiments,partitioning the plurality of cells comprises partitioning the pluralityof cells associated with the plurality of cellular component-bindingreagents and a plurality of barcoding particles comprising the barcodingparticle to the plurality of partitions, wherein the partition of theplurality of partitions comprises the single cell from the plurality ofcells associated with the cellular component-binding reagent and thebarcoding particle. In some embodiments, the plurality of cellularcomponent targets comprises a cell-surface protein, an intracellularprotein, a cell marker, a B-cell receptor, a T-cell receptor, anantibody, a major histocompatibility complex, a tumor antigen, areceptor, or a combination thereof. In some embodiments, the barcodingparticle is a Sepharose bead, a streptavidin bead, an agarose bead, amagnetic bead, a silica bead, a silica-like bead, a hydrogel bead, ananti-biotin microbead, an anti-fluorochrome microbead, or a hydrogelbead. In some embodiments, the partition is a well or a droplet. In someembodiments, the plurality of cells comprises T cells, B cells, tumorcells, myeloid cells, blood cells, normal cells, fetal cells, maternalcells, or a mixture thereof.

Some embodiments disclosed herein provide a composition comprising: aplurality of cellular component-binding reagents each associated with acellular component-binding reagent specific oligonucleotide comprising aunique identifier sequence for the cellular component-binding reagent,wherein the cellular component-binding reagent is capable ofspecifically binding to at least one of a plurality of cellularcomponent targets.

In some embodiments, the cellular component-binding reagent specificoligonucleotide comprises a first molecular label sequence. In someembodiments, the first molecular label sequence is 2-20 nucleotides inlength. In some embodiments, the first molecular label sequences of atleast two cellular component-binding reagent specific oligonucleotidesare different, and wherein the unique identifier sequences of the atleast two cellular component-binding reagent specific oligonucleotidesare identical. In some embodiments, the first molecular label sequencesof at least two cellular component-binding reagent specificoligonucleotides are different, and wherein the unique identifiersequences of the at least two cellular component-binding reagentspecific oligonucleotides are different. In some embodiments, thecellular component-binding reagent specific oligonucleotide comprisesthe sequence of a first universal primer, a complementary sequencethereof, a partial sequence thereof, or a combination thereof. In someembodiments, the first universal primer is 5-50 nucleotides in length.In some embodiments, the first universal primer comprises anamplification primer, a sequencing primer, or a combination thereof. Insome embodiments, the sequencing primer comprises a P7 sequencingprimer. In some embodiments, the cellular component-binding reagentspecific oligonucleotide comprises an alignment sequence adjacent to thepoly(dA) region. In some embodiments, the alignment sequence is one ormore nucleotides in length. In some embodiments, the alignment sequenceis two or more nucleotides in length. In some embodiments, the alignmentsequence comprises a guanine, a cytosine, a thymine, a uracil, or acombination thereof. In some embodiments, the alignment sequencecomprises a poly(dT) sequence, a poly(dG) sequence, a poly(dC) sequence,a poly(dU) sequence, or a combination thereof. In some embodiments, thealignment sequence is 5′ to the poly(dA) region.

In some embodiments, the cellular component-binding reagent specificoligonucleotide is associated with the cellular component-bindingreagent through a linker. In some embodiments, the linker comprises acarbon chain. In some embodiments, the carbon chain comprises 2-30carbons. In some embodiments, the carbon chain comprises 12 carbon. Insome embodiments, the linker comprises 5′ amino modifier C12 (5AmMC12),or a derivative thereof. In some embodiments, the cellularcomponent-binding reagent specific oligonucleotide is attached to thecellular component-binding reagent. In some embodiments, the cellularcomponent-binding reagent specific oligonucleotide is covalentlyattached to the cellular component-binding reagent. In some embodiments,the cellular component-binding reagent specific oligonucleotide isnon-covalently attached to the cellular component-binding reagent. Insome embodiments, the cellular component-binding reagent specificoligonucleotide is conjugated to the cellular component-binding reagent.In some embodiments, the cellular component-binding reagent specificoligonucleotide is conjugated to the cellular component-binding aptamerthrough a chemical group selected from the group consisting of a UVphotocleavable group, a streptavidin, a biotin, an amine, and acombination thereof.

In some embodiments, the plurality of cellular component-bindingreagents comprises a second cellular component-binding reagent. In someembodiments, the cellular component-binding reagent and the secondcellular component-binding reagent have at least 60%, 70%, 80%, 90%, or95% sequence identity. In some embodiments, the cellularcomponent-binding reagent and the second protein-binding reagent areidentical. In some embodiments, the cellular component-binding aptamerand the second cellular component-binding aptamer are different. In someembodiments, the protein targets, or the cellular component targets, ofthe cellular component-binding reagent and the second cellularcomponent-binding reagent are identical. In some embodiments, thecellular component-binding reagent and the second cellularcomponent-binding reagent are capable of binding to different regions ofa cellular component target. In some embodiments, the cellular componenttargets of the cellular component-binding reagent and the secondcellular component-binding reagent are different.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a non-limiting exemplary stochastic barcode.

FIG. 2 shows a non-limiting exemplary workflow of stochastic barcodingand digital counting.

FIG. 3 is a schematic illustration showing a non-limiting exemplaryprocess for generating an indexed library of the stochastically barcodedtargets from a plurality of targets.

FIG. 4 shows a schematic illustration of an exemplary protein bindingreagent (antibody illustrated here) associated with an oligonucleotidecomprising a unique identifier for the protein binding reagent.

FIG. 5 shows a schematic illustration of an exemplary binding reagent(antibody illustrated here) associated with an oligonucleotidecomprising a unique identifier for sample indexing to determine cellsfrom the same or different samples.

FIG. 6 shows a schematic illustration of an exemplary workflow of usingoligonucleotide-associated antibodies to determine cellular componentexpression (e.g., protein expression) and gene expression simultaneouslyin a high throughput manner.

FIG. 7 shows a schematic illustration of an exemplary workflow of usingoligonucleotide-associated antibodies for sample indexing.

FIG. 8 shows a schematic illustration of a non-limiting exemplaryworkflow of barcoding of a binding reagent oligonucleotide (antibodyoligonucleotide illustrated here) that is associated with a bindingreagent (antibody illustrated here).

FIGS. 9A-9D show non-limiting exemplary designs of oligonucleotides fordetermining protein expression and gene expression simultaneously andfor sample indexing.

FIG. 10 shows a schematic illustration of a non-limiting exemplaryoligonucleotide sequence for determining protein expression and geneexpression simultaneously and for sample indexing.

FIGS. 11A-11B show non-limiting exemplary designs of oligonucleotidesfor determining protein expression and gene expression simultaneouslyand for sample indexing.

DETAILED DESCRIPTION

In the following detailed description, reference is made to theaccompanying drawings, which form a part hereof. In the drawings,similar symbols typically identify similar components, unless contextdictates otherwise. The illustrative embodiments described in thedetailed description, drawings, and claims are not meant to be limiting.Other embodiments may be utilized, and other changes may be made,without departing from the spirit or scope of the subject matterpresented herein. It will be readily understood that the aspects of thepresent disclosure, as generally described herein, and illustrated inthe Figures, can be arranged, substituted, combined, separated, anddesigned in a wide variety of different configurations, all of which areexplicitly contemplated herein and made part of the disclosure herein.

All patents, published patent applications, other publications, andsequences from GenBank, and other databases referred to herein areincorporated by reference in their entirety with respect to the relatedtechnology.

Quantifying small numbers of nucleic acids, for example messengerribonucleotide acid (mRNA) molecules, is clinically important fordetermining, for example, the genes that are expressed in a cell atdifferent stages of development or under different environmentalconditions. However, it can also be very challenging to determine theabsolute number of nucleic acid molecules (e.g., mRNA molecules),especially when the number of molecules is very small. One method todetermine the absolute number of molecules in a sample is digitalpolymerase chain reaction (PCR). Ideally, PCR produces an identical copyof a molecule at each cycle. However, PCR can have disadvantages suchthat each molecule replicates with a stochastic probability, and thisprobability varies by PCR cycle and gene sequence, resulting inamplification bias and inaccurate gene expression measurements.Stochastic barcodes with unique molecular labels (also referred to asmolecular indexes (MIs)) can be used to count the number of moleculesand correct for amplification bias. Stochastic barcoding such as thePrecise™ assay (Cellular Research, Inc., Palo Alto, Calif.) can correctfor bias induced by PCR and library preparation steps by using molecularlabels (MLs) to label mRNAs during reverse transcription (RT).

The Precise™ assay can utilize a non-depleting pool of stochasticbarcodes with large number, for example 6561 to 65536, unique molecularlabels on poly(T) oligonucleotides to hybridize to all poly(A)-mRNAs ina sample during the RT step. A stochastic barcode can comprise auniversal PCR priming site. During RT, target gene molecules reactrandomly with stochastic barcodes. Each target molecule can hybridize toa stochastic barcode resulting to generate stochastically barcodedcomplementary ribonucleotide acid (cDNA) molecules). After labeling,stochastically barcoded cDNA molecules from microwells of a microwellplate can be pooled into a single tube for PCR amplification andsequencing. Raw sequencing data can be analyzed to produce the number ofreads, the number of stochastic barcodes with unique molecular labels,and the numbers of mRNA molecules.

Methods for determining mRNA expression profiles of single cells can beperformed in a massively parallel manner. For example, the Precise™assay can be used to determine the mRNA expression profiles of more than10000 cells simultaneously. The number of single cells (e.g., 100s or1000s of singles) for analysis per sample can be lower than the capacityof the current single cell technology. Pooling of cells from differentsamples enables improved utilization of the capacity of the currentsingle technology, thus lowering reagents wasted and the cost of singlecell analysis. The disclosure provides methods of sample indexing fordistinguishing cells of different samples for cDNA library preparationfor cell analysis, such as single cell analysis. Pooling of cells fromdifferent samples can minimize the variations in cDNA librarypreparation of cells of different samples, thus enabling more accuratecomparisons of different samples.

Disclosed herein include methods for sample identification. In someembodiments, the method comprises: contacting each of a plurality ofsamples with a sample indexing composition of a plurality of sampleindexing compositions, respectively, wherein each of the plurality ofsamples comprises one or more cells each comprising one or more cellularcomponent targets, wherein the sample indexing composition comprises acellular component-binding reagent associated with a sample indexingoligonucleotide, wherein the cellular component-binding reagent iscapable of specifically binding to at least one of the one or morecellular component targets, wherein the sample indexing oligonucleotidecomprises a sample indexing sequence, and wherein sample indexingsequences of at least two sample indexing compositions of the pluralityof sample indexing compositions comprise different sequences; andidentifying sample origin of at least one cell of the one or more cellsbased on the sample indexing sequence of at least one sample indexingoligonucleotide of the plurality of sample indexing compositions.

Disclosed herein include methods for measuring cellular componentexpression in cells. In some embodiments, the method comprises:contacting a plurality of cellular component-binding reagents with aplurality of cells comprising a plurality of cellular component targets,wherein each of the plurality of cellular component-binding reagentscomprises a cellular component-binding reagent specific oligonucleotidecomprising a unique identifier sequence for the cellularcomponent-binding reagent, and wherein the cellular component-bindingreagent is capable of specifically binding to at least one of theplurality of cellular component targets; extending barcodes hybridizedto the cellular component-binding reagent specific oligonucleotides, orproducts thereof, to produce a plurality of labeled nucleic acids,wherein each of the labeled nucleic acid comprises a unique identifiersequence, or a complementary sequence thereof, and a first molecularlabel sequence, or a complementary sequence thereof; and obtainingsequence information of the plurality of labeled nucleic acids, acomplementary sequence thereof, or a portion thereof to determine thenumber of copies of at least one cellular component target of theplurality of cellular component targets in one or more of the pluralityof cells.

Disclosed herein is a plurality of sample indexing compositions. Each ofthe plurality of sample indexing compositions can comprise a cellularcomponent-binding reagent associated with a sample indexingoligonucleotide, wherein the cellular component-binding reagent iscapable of specifically binding to at least one cellular componenttarget, wherein the sample indexing oligonucleotide comprises a sampleindexing sequence for identifying sample origin of one or more cells ofa sample, and wherein sample indexing sequences of at least two sampleindexing compositions of the plurality of sample indexing compositionscomprise different sequences.

In some embodiments, the composition comprises: a plurality of cellularcomponent-binding reagents each associated with a cellularcomponent-binding reagent specific oligonucleotide comprising a uniqueidentifier sequence for the cellular component-binding reagent, whereinthe cellular component-binding reagent is capable of specificallybinding to at least one of a plurality of cellular component targets.

Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the present disclosure belongs. See, e.g., Singleton etal., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley& Sons (New York, N.Y. 1994); Sambrook et al., Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, N.Y.1989). For purposes of the present disclosure, the following terms aredefined below.

As used herein, the term “adaptor” can mean a sequence to facilitateamplification or sequencing of associated nucleic acids. The associatednucleic acids can comprise target nucleic acids. The associated nucleicacids can comprise one or more of spatial labels, target labels, samplelabels, indexing label, or barcode sequences (e.g., molecular labels).The adapters can be linear. The adaptors can be pre-adenylated adapters.The adaptors can be double- or single-stranded. One or more adaptor canbe located on the 5′ or 3′ end of a nucleic acid. When the adaptorscomprise known sequences on the 5′ and 3′ ends, the known sequences canbe the same or different sequences. An adaptor located on the 5′ and/or3′ ends of a polynucleotide can be capable of hybridizing to one or moreoligonucleotides immobilized on a surface. An adapter can, in someembodiments, comprise a universal sequence. A universal sequence can bea region of nucleotide sequence that is common to two or more nucleicacid molecules. The two or more nucleic acid molecules can also haveregions of different sequence. Thus, for example, the 5′ adapters cancomprise identical and/or universal nucleic acid sequences and the 3′adapters can comprise identical and/or universal sequences. A universalsequence that may be present in different members of a plurality ofnucleic acid molecules can allow the replication or amplification ofmultiple different sequences using a single universal primer that iscomplementary to the universal sequence. Similarly, at least one, two(e.g., a pair) or more universal sequences that may be present indifferent members of a collection of nucleic acid molecules can allowthe replication or amplification of multiple different sequences usingat least one, two (e.g., a pair) or more single universal primers thatare complementary to the universal sequences. Thus, a universal primerincludes a sequence that can hybridize to such a universal sequence. Thetarget nucleic acid sequence-bearing molecules may be modified to attachuniversal adapters (e.g., non-target nucleic acid sequences) to one orboth ends of the different target nucleic acid sequences. The one ormore universal primers attached to the target nucleic acid can providesites for hybridization of universal primers. The one or more universalprimers attached to the target nucleic acid can be the same or differentfrom each other.

As used herein, an antibody can be a full-length (e.g., naturallyoccurring or formed by normal immunoglobulin gene fragmentrecombinatorial processes) immunoglobulin molecule (e.g., an IgGantibody) or an immunologically active (i.e., specifically binding)portion of an immunoglobulin molecule, like an antibody fragment.

In some embodiments, an antibody is a functional antibody fragment. Forexample, an antibody fragment can be a portion of an antibody such asF(ab′)2, Fab′, Fab, Fv, sFv and the like. An antibody fragment can bindwith the same antigen that is recognized by the full-length antibody. Anantibody fragment can include isolated fragments consisting of thevariable regions of antibodies, such as the “Fv” fragments consisting ofthe variable regions of the heavy and light chains and recombinantsingle chain polypeptide molecules in which light and heavy variableregions are connected by a peptide linker (“scFv proteins”). Exemplaryantibodies can include, but are not limited to, antibodies for cancercells, antibodies for viruses, antibodies that bind to cell surfacereceptors (for example, CD8, CD34, and CD45), and therapeuticantibodies.

As used herein the term “associated” or “associated with” can mean thattwo or more species are identifiable as being co-located at a point intime. An association can mean that two or more species are or werewithin a similar container. An association can be an informaticsassociation. For example, digital information regarding two or morespecies can be stored and can be used to determine that one or more ofthe species were co-located at a point in time. An association can alsobe a physical association. In some embodiments, two or more associatedspecies are “tethered”, “attached”, or “immobilized” to one another orto a common solid or semisolid surface. An association may refer tocovalent or non-covalent means for attaching labels to solid orsemi-solid supports such as beads. An association may be a covalent bondbetween a target and a label. An association can comprise hybridizationbetween two molecules (such as a target molecule and a label).

As used herein, the term “complementary” can refer to the capacity forprecise pairing between two nucleotides. For example, if a nucleotide ata given position of a nucleic acid is capable of hydrogen bonding with anucleotide of another nucleic acid, then the two nucleic acids areconsidered to be complementary to one another at that position.Complementarity between two single-stranded nucleic acid molecules maybe “partial,” in which only some of the nucleotides bind, or it may becomplete when total complementarity exists between the single-strandedmolecules. A first nucleotide sequence can be said to be the“complement” of a second sequence if the first nucleotide sequence iscomplementary to the second nucleotide sequence. A first nucleotidesequence can be said to be the “reverse complement” of a secondsequence, if the first nucleotide sequence is complementary to asequence that is the reverse (i.e., the order of the nucleotides isreversed) of the second sequence. As used herein, the terms“complement”, “complementary”, and “reverse complement” can be usedinterchangeably. It is understood from the disclosure that if a moleculecan hybridize to another molecule it may be the complement of themolecule that is hybridizing.

As used herein, the term “digital counting” can refer to a method forestimating a number of target molecules in a sample. Digital countingcan include the step of determining a number of unique labels that havebeen associated with targets in a sample. This methodology, which can bestochastic in nature, transforms the problem of counting molecules fromone of locating and identifying identical molecules to a series ofyes/no digital questions regarding detection of a set of predefinedlabels.

As used herein, the term “label” or “labels” can refer to nucleic acidcodes associated with a target within a sample. A label can be, forexample, a nucleic acid label. A label can be an entirely or partiallyamplifiable label. A label can be entirely or partially sequencablelabel. A label can be a portion of a native nucleic acid that isidentifiable as distinct. A label can be a known sequence. A label cancomprise a junction of nucleic acid sequences, for example a junction ofa native and non-native sequence. As used herein, the term “label” canbe used interchangeably with the terms, “index”, “tag,” or “label-tag.”Labels can convey information. For example, in various embodiments,labels can be used to determine an identity of a sample, a source of asample, an identity of a cell, and/or a target.

As used herein, the term “non-depleting reservoirs” can refer to a poolof barcodes (e.g., stochastic barcodes) made up of many differentlabels. A non-depleting reservoir can comprise large numbers ofdifferent barcodes such that when the non-depleting reservoir isassociated with a pool of targets each target is likely to be associatedwith a unique barcode. The uniqueness of each labeled target moleculecan be determined by the statistics of random choices, and depends onthe number of copies of identical target molecules in the collectioncompared to the diversity of labels. The size of the resulting set oflabeled target molecules can be determined by the stochastic nature ofthe barcoding process, and analysis of the number of barcodes detectedthen allows calculation of the number of target molecules present in theoriginal collection or sample. When the ratio of the number of copies ofa target molecule present to the number of unique barcodes is low, thelabeled target molecules are highly unique (i.e., there is a very lowprobability that more than one target molecule will have been labeledwith a given label).

As used herein, the term “nucleic acid” refers to a polynucleotidesequence, or fragment thereof. A nucleic acid can comprise nucleotides.A nucleic acid can be exogenous or endogenous to a cell. A nucleic acidcan exist in a cell-free environment. A nucleic acid can be a gene orfragment thereof. A nucleic acid can be DNA. A nucleic acid can be RNA.A nucleic acid can comprise one or more analogs (e.g., altered backbone,sugar, or nucleobase). Some non-limiting examples of analogs include:5-bromouracil, peptide nucleic acid, xeno nucleic acid, morpholinos,locked nucleic acids, glycol nucleic acids, threose nucleic acids,dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g.,rhodamine or fluorescein linked to the sugar), thiol containingnucleotides, biotin linked nucleotides, fluorescent base analogs, CpGislands, methyl-7-guanosine, methylated nucleotides, inosine,thiouridine, pseudouridine, dihydrouridine, queuosine, and wyosine.“Nucleic acid”, “polynucleotide, “target polynucleotide”, and “targetnucleic acid” can be used interchangeably.

A nucleic acid can comprise one or more modifications (e.g., a basemodification, a backbone modification), to provide the nucleic acid witha new or enhanced feature (e.g., improved stability). A nucleic acid cancomprise a nucleic acid affinity tag. A nucleoside can be a base-sugarcombination. The base portion of the nucleoside can be a heterocyclicbase. The two most common classes of such heterocyclic bases are thepurines and the pyrimidines. Nucleotides can be nucleosides that furtherinclude a phosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to the 2′, the 3′, or the 5′ hydroxylmoiety of the sugar. In forming nucleic acids, the phosphate groups cancovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn, the respective ends of this linearpolymeric compound can be further joined to form a circular compound;however, linear compounds are generally suitable. In addition, linearcompounds may have internal nucleotide base complementarity and maytherefore fold in a manner as to produce a fully or partiallydouble-stranded compound. Within nucleic acids, the phosphate groups cancommonly be referred to as forming the internucleoside backbone of thenucleic acid. The linkage or backbone can be a 3′ to 5′ phosphodiesterlinkage.

A nucleic acid can comprise a modified backbone and/or modifiedinternucleoside linkages. Modified backbones can include those thatretain a phosphorus atom in the backbone and those that do not have aphosphorus atom in the backbone. Suitable modified nucleic acidbackbones containing a phosphorus atom therein can include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonate such as 3′-alkylene phosphonates, 5′-alkylene phosphonates,chiral phosphonates, phosphinates, phosphoramidates including 3′-aminophosphoramidate and aminoalkyl phosphoramidates, phosphorodiamidates,thionophosphoramidates, thionoalkylphosphonats,thionoalkylphosphotriesters, selenophosphates, and boranophosphateshaving normal 3′-5′ linkages, 2′-5′ linked analogs, and those havinginverted polarity wherein one or more internucleotide linkages is a 3′to 3′, a 5′ to 5′ or a 2′ to 2′ linkage.

A nucleic acid can comprise polynucleotide backbones that are formed byshort chain alkyl or cycloalkyl internucleoside linkages, mixedheteroatom and alkyl or cycloalkyl internucleoside linkages, or one ormore short chain heteroatomic or heterocyclic internucleoside linkages.These can include those having morpholino linkages (formed in part fromthe sugar portion of a nucleoside); siloxane backbones; sulfide,sulfoxide and sulfone backbones; formacetyl and thioformacetylbackbones; methylene formacetyl and thioformacetyl backbones; riboacetylbackbones; alkene containing backbones; sulfamate backbones;methyleneimino and methylenehydrazino backbones; sulfonate andsulfonamide backbones; amide backbones; and others having mixed N, O, Sand CH₂ component parts.

A nucleic acid can comprise a nucleic acid mimetic. The term “mimetic”can be intended to include polynucleotides wherein only the furanosering or both the furanose ring and the internucleotide linkage arereplaced with non-furanose groups, replacement of only the furanose ringcan also be referred as being a sugar surrogate. The heterocyclic basemoiety or a modified heterocyclic base moiety can be maintained forhybridization with an appropriate target nucleic acid. One such nucleicacid can be a peptide nucleic acid (PNA). In a PNA, the sugar-backboneof a polynucleotide can be replaced with an amide containing backbone,in particular an aminoethylglycine backbone. The nucleotides can beretained and are bound directly or indirectly to aza nitrogen atoms ofthe amide portion of the backbone. The backbone in PNA compounds cancomprise two or more linked aminoethylglycine units which gives PNA anamide containing backbone. The heterocyclic base moieties can be bounddirectly or indirectly to aza nitrogen atoms of the amide portion of thebackbone.

A nucleic acid can comprise a morpholino backbone structure. Forexample, a nucleic acid can comprise a 6-membered morpholino ring inplace of a ribose ring. In some of these embodiments, aphosphorodiamidate or other non-phosphodiester internucleoside linkagecan replace a phosphodiester linkage.

A nucleic acid can comprise linked morpholino units (e.g., morpholinonucleic acid) having heterocyclic bases attached to the morpholino ring.Linking groups can link the morpholino monomeric units in a morpholinonucleic acid. Non-ionic morpholino-based oligomeric compounds can haveless undesired interactions with cellular proteins. Morpholino-basedpolynucleotides can be nonionic mimics of nucleic acids. A variety ofcompounds within the morpholino class can be joined using differentlinking groups. A further class of polynucleotide mimetic can bereferred to as cyclohexenyl nucleic acids (CeNA). The furanose ringnormally present in a nucleic acid molecule can be replaced with acyclohexenyl ring. CeNA DMT protected phosphoramidite monomers can beprepared and used for oligomeric compound synthesis usingphosphoramidite chemistry. The incorporation of CeNA monomers into anucleic acid chain can increase the stability of a DNA/RNA hybrid. CeNAoligoadenylates can form complexes with nucleic acid complements withsimilar stability to the native complexes. A further modification caninclude Locked Nucleic Acids (LNAs) in which the 2′-hydroxyl group islinked to the 4′ carbon atom of the sugar ring thereby forming a 2′-C,4′-C-oxymethylene linkage thereby forming a bicyclic sugar moiety. Thelinkage can be a methylene (—CH₂), group bridging the 2′ oxygen atom andthe 4′ carbon atom wherein n is 1 or 2. LNA and LNA analogs can displayvery high duplex thermal stabilities with complementary nucleic acid(Tm=+3 to +10° C.), stability towards 3′-exonucleolytic degradation andgood solubility properties.

A nucleic acid may also include nucleobase (often referred to simply as“base”) modifications or substitutions. As used herein, “unmodified” or“natural” nucleobases can include the purine bases, (e.g., adenine (A)and guanine (G)), and the pyrimidine bases, (e.g., thymine (T), cytosine(C) and uracil (U)). Modified nucleobases can include other syntheticand natural nucleobases such as 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl(—C═C—CH3) uracil and cytosine and other alkynyl derivatives ofpyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Modifiednucleobases can include tricyclic pyrimidines such as phenoxazinecytidine(1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.,9-(2-aminoethoxy)-H-pyrimido(5,4-(b) (1,4)benzoxazin-2(3H)-one),phenothiazine cytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one),G-clamps such as a substituted phenoxazine cytidine (e.g.,9-(2-aminoethoxy)-H-pyrimido(5,4-(b) (1,4)benzoxazin-2(3H)-one),carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindolecytidine (H-pyrido(3′,2′:4,5)pyrrolo[2,3-d]pyrimidin-2-one).

As used herein, the term “sample” can refer to a composition comprisingtargets. Suitable samples for analysis by the disclosed methods,devices, and systems include cells, tissues, organs, or organisms.

As used herein, the term “sampling device” or “device” can refer to adevice which may take a section of a sample and/or place the section ona substrate. A sample device can refer to, for example, a fluorescenceactivated cell sorting (FACS) machine, a cell sorter machine, a biopsyneedle, a biopsy device, a tissue sectioning device, a microfluidicdevice, a blade grid, and/or a microtome.

As used herein, the term “solid support” can refer to discrete solid orsemi-solid surfaces to which a plurality of barcodes (e.g., stochasticbarcodes) may be attached. A solid support may encompass any type ofsolid, porous, or hollow sphere, ball, bearing, cylinder, or othersimilar configuration composed of plastic, ceramic, metal, or polymericmaterial (e.g., hydrogel) onto which a nucleic acid may be immobilized(e.g., covalently or non-covalently). A solid support may comprise adiscrete particle that may be spherical (e.g., microspheres) or have anon-spherical or irregular shape, such as cubic, cuboid, pyramidal,cylindrical, conical, oblong, or disc-shaped, and the like. A bead canbe non-spherical in shape. A plurality of solid supports spaced in anarray may not comprise a substrate. A solid support may be usedinterchangeably with the term “bead.”

As used herein, the term “stochastic barcode” can refer to apolynucleotide sequence comprising labels of the present disclosure. Astochastic barcode can be a polynucleotide sequence that can be used forstochastic barcoding. Stochastic barcodes can be used to quantifytargets within a sample. Stochastic barcodes can be used to control forerrors which may occur after a label is associated with a target. Forexample, a stochastic barcode can be used to assess amplification orsequencing errors. A stochastic barcode associated with a target can becalled a stochastic barcode-target or stochastic barcode-tag-target.

As used herein, the term “gene-specific stochastic barcode” can refer toa polynucleotide sequence comprising labels and a target-binding regionthat is gene-specific. A stochastic barcode can be a polynucleotidesequence that can be used for stochastic barcoding. Stochastic barcodescan be used to quantify targets within a sample. Stochastic barcodes canbe used to control for errors which may occur after a label isassociated with a target. For example, a stochastic barcode can be usedto assess amplification or sequencing errors. A stochastic barcodeassociated with a target can be called a stochastic barcode-target orstochastic barcode-tag-target.

As used herein, the term “stochastic barcoding” can refer to the randomlabeling (e.g., barcoding) of nucleic acids. Stochastic barcoding canutilize a recursive Poisson strategy to associate and quantify labelsassociated with targets. As used herein, the term “stochastic barcoding”can be used interchangeably with “stochastic labeling.”

As used here, the term “target” can refer to a composition which can beassociated with a barcode (e.g., a stochastic barcode). Exemplarysuitable targets for analysis by the disclosed methods, devices, andsystems include oligonucleotides, DNA, RNA, mRNA, microRNA, tRNA, andthe like. Targets can be single or double stranded. In some embodiments,targets can be proteins, peptides, or polypeptides. In some embodiments,targets are lipids. As used herein, “target” can be used interchangeablywith “species.”

As used herein, the term “reverse transcriptases” can refer to a groupof enzymes having reverse transcriptase activity (i.e., that catalyzesynthesis of DNA from an RNA template). In general, such enzymesinclude, but are not limited to, retroviral reverse transcriptase,retrotransposon reverse transcriptase, retroplasmid reversetranscriptases, retron reverse transcriptases, bacterial reversetranscriptases, group II intron-derived reverse transcriptase, andmutants, variants or derivatives thereof. Non-retroviral reversetranscriptases include non-LTR retrotransposon reverse transcriptases,retroplasmid reverse transcriptases, retron reverse transcriptases, andgroup II intron reverse transcriptases. Examples of group II intronreverse transcriptases include the Lactococcus lactis LI.LtrB intronreverse transcriptase, the Thermosynechococcus elongatus TeI4c intronreverse transcriptase, or the Geobacillus stearothermophilus GsI-IICintron reverse transcriptase. Other classes of reverse transcriptasescan include many classes of non-retroviral reverse transcriptases (i.e.,retrons, group II introns, and diversity-generating retroelements amongothers).

The terms “universal adaptor primer,” “universal primer adaptor” or“universal adaptor sequence” are used interchangeably to refer to anucleotide sequence that can be used to hybridize to barcodes (e.g.,stochastic barcodes) to generate gene-specific barcodes. A universaladaptor sequence can, for example, be a known sequence that is universalacross all barcodes used in methods of the disclosure. For example, whenmultiple targets are being labeled using the methods disclosed herein,each of the target-specific sequences may be linked to the sameuniversal adaptor sequence. In some embodiments, more than one universaladaptor sequences may be used in the methods disclosed herein. Forexample, when multiple targets are being labeled using the methodsdisclosed herein, at least two of the target-specific sequences arelinked to different universal adaptor sequences. A universal adaptorprimer and its complement may be included in two oligonucleotides, oneof which comprises a target-specific sequence and the other comprises abarcode. For example, a universal adaptor sequence may be part of anoligonucleotide comprising a target-specific sequence to generate anucleotide sequence that is complementary to a target nucleic acid. Asecond oligonucleotide comprising a barcode and a complementary sequenceof the universal adaptor sequence may hybridize with the nucleotidesequence and generate a target-specific barcode (e.g., a target-specificstochastic barcode). In some embodiments, a universal adaptor primer hasa sequence that is different from a universal PCR primer used in themethods of this disclosure.

Barcodes

Barcoding, such as stochastic barcoding, has been described in, forexample, Fu et al., Proc Natl Acad Sci U.S.A., 2011 May 31,108(22):9026-31; U.S. Patent Application Publication No. US2011/0160078;Fan et al., Science, 2015 Feb. 6, 347(6222):1258367; US PatentApplication Publication No. US2015/0299784; and PCT ApplicationPublication No. WO2015/031691; the content of each of these, includingany supporting or supplemental information or material, is incorporatedherein by reference in its entirety. In some embodiments, the barcodedisclosed herein can be a stochastic barcode which can be apolynucleotide sequence that may be used to stochastically label (e.g.,barcode, tag) a target. Barcodes can be referred to stochastic barcodesif the ratio of the number of different barcode sequences of thestochastic barcodes and the number of occurrence of any of the targetsto be labeled can be, or be about, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1,8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1,20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, or a number or arange between any two of these values. A target can be an mRNA speciescomprising mRNA molecules with identical or nearly identical sequences.Barcodes can be referred to as stochastic barcodes if the ratio of thenumber of different barcode sequences of the stochastic barcodes and thenumber of occurrence of any of the targets to be labeled is at least, oris at most, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1,12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30:1, 40:1, 50:1,60:1, 70:1, 80:1, 90:1, or 100:1. Barcode sequences of stochasticbarcodes can be referred to as molecular labels.

A barcode, for example a stochastic barcode, can comprise one or morelabels. Exemplary labels can include a universal label, a cell label, abarcode sequence (e.g., a molecular label), a sample label, a platelabel, a spatial label, and/or a pre-spatial label. FIG. 1 illustratesan exemplary barcode 104 with a spatial label. The barcode 104 cancomprise a 5′amine that may link the barcode to a solid support 108. Thebarcode can comprise a universal label, a dimension label, a spatiallabel, a cell label, and/or a molecular label. The order of differentlabels (including but not limited to the universal label, the dimensionlabel, the spatial label, the cell label, and the molecule label) in thebarcode can vary. For example, as shown in FIG. 1, the universal labelmay be the 5′-most label, and the molecular label may be the 3′-mostlabel. The spatial label, dimension label, and the cell label may be inany order. In some embodiments, the universal label, the spatial label,the dimension label, the cell label, and the molecular label are in anyorder. The barcode can comprise a target-binding region. Thetarget-binding region can interact with a target (e.g., target nucleicacid, RNA, mRNA, DNA) in a sample. For example, a target-binding regioncan comprise an oligo(dT) sequence which can interact with poly(A) tailsof mRNAs. In some instances, the labels of the barcode (e.g., universallabel, dimension label, spatial label, cell label, and barcode sequence)may be separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, or 20 or more nucleotides.

A label, for example the cell label, can comprise a unique set ofnucleic acid sub-sequences of defined length, e.g., seven nucleotideseach (equivalent to the number of bits used in some Hamming errorcorrection codes), which can be designed to provide error correctioncapability. The set of error correction sub-sequences comprise sevennucleotide sequences can be designed such that any pairwise combinationof sequences in the set exhibits a defined “genetic distance” (or numberof mismatched bases), for example, a set of error correctionsub-sequences can be designed to exhibit a genetic distance of threenucleotides. In this case, review of the error correction sequences inthe set of sequence data for labeled target nucleic acid molecules(described more fully below) can allow one to detect or correctamplification or sequencing errors. In some embodiments, the length ofthe nucleic acid sub-sequences used for creating error correction codescan vary, for example, they can be, or be about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 30, 31, 40, 50, or a number or a range between any two ofthese values, nucleotides in length. In some embodiments, nucleic acidsub-sequences of other lengths can be used for creating error correctioncodes.

The barcode can comprise a target-binding region. The target-bindingregion can interact with a target in a sample. The target can be, orcomprise, ribonucleic acids (RNAs), messenger RNAs (mRNAs), microRNAs,small interfering RNAs (siRNAs), RNA degradation products, RNAs eachcomprising a poly(A) tail, or any combination thereof. In someembodiments, the plurality of targets can include deoxyribonucleic acids(DNAs).

In some embodiments, a target-binding region can comprise an oligo(dT)sequence which can interact with poly(A) tails of mRNAs. One or more ofthe labels of the barcode (e.g., the universal label, the dimensionlabel, the spatial label, the cell label, and the barcode sequences(e.g., molecular label)) can be separated by a spacer from another oneor two of the remaining labels of the barcode. The spacer can be, forexample, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20, or more nucleotides. In some embodiments, none of the labelsof the barcode is separated by spacer.

Universal Labels

A barcode can comprise one or more universal labels. In someembodiments, the one or more universal labels can be the same for allbarcodes in the set of barcodes attached to a given solid support. Insome embodiments, the one or more universal labels can be the same forall barcodes attached to a plurality of beads. In some embodiments, auniversal label can comprise a nucleic acid sequence that is capable ofhybridizing to a sequencing primer. Sequencing primers can be used forsequencing barcodes comprising a universal label. Sequencing primers(e.g., universal sequencing primers) can comprise sequencing primersassociated with high-throughput sequencing platforms. In someembodiments, a universal label can comprise a nucleic acid sequence thatis capable of hybridizing to a PCR primer. In some embodiments, theuniversal label can comprise a nucleic acid sequence that is capable ofhybridizing to a sequencing primer and a PCR primer. The nucleic acidsequence of the universal label that is capable of hybridizing to asequencing or PCR primer can be referred to as a primer binding site. Auniversal label can comprise a sequence that can be used to initiatetranscription of the barcode. A universal label can comprise a sequencethat can be used for extension of the barcode or a region within thebarcode. A universal label can be, or be about, 1, 2, 3, 4, 5, 10, 15,20, 25, 30, 35, 40, 45, 50, or a number or a range between any two ofthese values, nucleotides in length. For example, a universal label cancomprise at least about 10 nucleotides. A universal label can be atleast, or be at most, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,100, 200, or 300 nucleotides in length. In some embodiments, a cleavablelinker or modified nucleotide can be part of the universal labelsequence to enable the barcode to be cleaved off from the support.

Dimension Labels

A barcode can comprise one or more dimension labels. In someembodiments, a dimension label can comprise a nucleic acid sequence thatprovides information about a dimension in which the labeling (e.g.,stochastic labeling) occurred. For example, a dimension label canprovide information about the time at which a target was barcoded. Adimension label can be associated with a time of barcoding (e.g.,stochastic barcoding) in a sample. A dimension label can be activated atthe time of labeling. Different dimension labels can be activated atdifferent times. The dimension label provides information about theorder in which targets, groups of targets, and/or samples were barcoded.For example, a population of cells can be barcoded at the G0 phase ofthe cell cycle. The cells can be pulsed again with barcodes (e.g.,stochastic barcodes) at the G1 phase of the cell cycle. The cells can bepulsed again with barcodes at the S phase of the cell cycle, and so on.Barcodes at each pulse (e.g., each phase of the cell cycle), cancomprise different dimension labels. In this way, the dimension labelprovides information about which targets were labelled at which phase ofthe cell cycle. Dimension labels can interrogate many differentbiological times. Exemplary biological times can include, but are notlimited to, the cell cycle, transcription (e.g., transcriptioninitiation), and transcript degradation. In another example, a sample(e.g., a cell, a population of cells) can be labeled before and/or aftertreatment with a drug and/or therapy. The changes in the number ofcopies of distinct targets can be indicative of the sample's response tothe drug and/or therapy.

A dimension label can be activatable. An activatable dimension label canbe activated at a specific time point. The activatable label can be, forexample, constitutively activated (e.g., not turned off). Theactivatable dimension label can be, for example, reversibly activated(e.g., the activatable dimension label can be turned on and turned off).The dimension label can be, for example, reversibly activatable at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. The dimension label can bereversibly activatable, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9,10 or more times. In some embodiments, the dimension label can beactivated with fluorescence, light, a chemical event (e.g., cleavage,ligation of another molecule, addition of modifications (e.g.,pegylated, sumoylated, acetylated, methylated, deacetylated,demethylated), a photochemical event (e.g., photocaging), andintroduction of a non-natural nucleotide.

The dimension label can, in some embodiments, be identical for allbarcodes (e.g., stochastic barcodes) attached to a given solid support(e.g., a bead), but different for different solid supports (e.g.,beads). In some embodiments, at least 60%, 70%, 80%, 85%, 90%, 95%, 97%,99% or 100%, of barcodes on the same solid support can comprise the samedimension label. In some embodiments, at least 60% of barcodes on thesame solid support can comprise the same dimension label. In someembodiments, at least 95% of barcodes on the same solid support cancomprise the same dimension label.

There can be as many as 10⁶ or more unique dimension label sequencesrepresented in a plurality of solid supports (e.g., beads). A dimensionlabel can be, or be about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45,50, or a number or a range between any two of these values, nucleotidesin length. A dimension label can be at least, or be at most, 1, 2, 3, 4,5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300, nucleotides inlength. A dimension label can comprise between about 5 to about 200nucleotides. A dimension label can comprise between about 10 to about150 nucleotides. A dimension label can comprise between about 20 toabout 125 nucleotides in length.

Spatial Labels

A barcode can comprise one or more spatial labels. In some embodiments,a spatial label can comprise a nucleic acid sequence that providesinformation about the spatial orientation of a target molecule which isassociated with the barcode. A spatial label can be associated with acoordinate in a sample. The coordinate can be a fixed coordinate. Forexample, a coordinate can be fixed in reference to a substrate. Aspatial label can be in reference to a two or three-dimensional grid. Acoordinate can be fixed in reference to a landmark. The landmark can beidentifiable in space. A landmark can be a structure which can beimaged. A landmark can be a biological structure, for example ananatomical landmark. A landmark can be a cellular landmark, for instancean organelle. A landmark can be a non-natural landmark such as astructure with an identifiable identifier such as a color code, barcode, magnetic property, fluorescents, radioactivity, or a unique sizeor shape. A spatial label can be associated with a physical partition(e.g., a well, a container, or a droplet). In some embodiments, multiplespatial labels are used together to encode one or more positions inspace.

The spatial label can be identical for all barcodes attached to a givensolid support (e.g., a bead), but different for different solid supports(e.g., beads). In some embodiments, the percentage of barcodes on thesame solid support comprising the same spatial label can be, or beabout, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, 100%, or a number or arange between any two of these values. In some embodiments, thepercentage of barcodes on the same solid support comprising the samespatial label can be at least, or be at most, 60%, 70%, 80%, 85%, 90%,95%, 97%, 99%, or 100%. In some embodiments, at least 60% of barcodes onthe same solid support can comprise the same spatial label. In someembodiments, at least 95% of barcodes on the same solid support cancomprise the same spatial label.

There can be as many as 10⁶ or more unique spatial label sequencesrepresented in a plurality of solid supports (e.g., beads). A spatiallabel can be, or be about, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40,45, 50, or a number or a range between any two of these values,nucleotides in length. A spatial label can be at least or at most 1, 2,3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300nucleotides in length. A spatial label can comprise between about 5 toabout 200 nucleotides. A spatial label can comprise between about 10 toabout 150 nucleotides. A spatial label can comprise between about 20 toabout 125 nucleotides in length.

Cell Labels

A barcode (e.g., a stochastic barcode) can comprise one or more celllabels. In some embodiments, a cell label can comprise a nucleic acidsequence that provides information for determining which target nucleicacid originated from which cell. In some embodiments, the cell label isidentical for all barcodes attached to a given solid support (e.g., abead), but different for different solid supports (e.g., beads). In someembodiments, the percentage of barcodes on the same solid supportcomprising the same cell label can be, or be about 60%, 70%, 80%, 85%,90%, 95%, 97%, 99%, 100%, or a number or a range between any two ofthese values. In some embodiments, the percentage of barcodes on thesame solid support comprising the same cell label can be, or be about60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or 100%. For example, at least60% of barcodes on the same solid support can comprise the same celllabel. As another example, at least 95% of barcodes on the same solidsupport can comprise the same cell label.

There can be as many as 10⁶ or more unique cell label sequencesrepresented in a plurality of solid supports (e.g., beads). A cell labelcan be, or be about, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,or a number or a range between any two of these values, nucleotides inlength. A cell label can be at least, or be at most, 1, 2, 3, 4, 5, 10,15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides in length.For example, a cell label can comprise between about 5 to about 200nucleotides. As another example, a cell label can comprise between about10 to about 150 nucleotides. As yet another example, a cell label cancomprise between about 20 to about 125 nucleotides in length.

Barcode Sequences

A barcode can comprise one or more barcode sequences. In someembodiments, a barcode sequence can comprise a nucleic acid sequencethat provides identifying information for the specific type of targetnucleic acid species hybridized to the barcode. A barcode sequence cancomprise a nucleic acid sequence that provides a counter (e.g., thatprovides a rough approximation) for the specific occurrence of thetarget nucleic acid species hybridized to the barcode (e.g.,target-binding region).

In some embodiments, a diverse set of barcode sequences are attached toa given solid support (e.g., a bead). In some embodiments, there can be,or be about, 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, or a number or arange between any two of these values, unique molecular label sequences.For example, a plurality of barcodes can comprise about 6561 barcodessequences with distinct sequences. As another example, a plurality ofbarcodes can comprise about 65536 barcode sequences with distinctsequences. In some embodiments, there can be at least, or be at most,10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, or 10⁹, unique barcode sequences. Theunique molecular label sequences can be attached to a given solidsupport (e.g., a bead).

The length of a barcode can be different in different implementations.For example, a barcode can be, or be about, 1, 2, 3, 4, 5, 10, 15, 20,25, 30, 35, 40, 45, 50, or a number or a range between any two of thesevalues, nucleotides in length. As another example, a barcode can be atleast, or be at most, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,100, 200, or 300 nucleotides in length.

Molecular Labels

A barcode (e.g., a stochastic barcode) can comprise one or moremolecular labels. Molecular labels can include barcode sequences. Insome embodiments, a molecular label can comprise a nucleic acid sequencethat provides identifying information for the specific type of targetnucleic acid species hybridized to the barcode. A molecular label cancomprise a nucleic acid sequence that provides a counter for thespecific occurrence of the target nucleic acid species hybridized to thebarcode (e.g., target-binding region).

In some embodiments, a diverse set of molecular labels are attached to agiven solid support (e.g., a bead). In some embodiments, there can be,or be about, 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, or a number or arange between any two of these values, of unique molecular labelsequences. For example, a plurality of barcodes can comprise about 6561molecular labels with distinct sequences. As another example, aplurality of barcodes can comprise about 65536 molecular labels withdistinct sequences. In some embodiments, there can be at least, or be atmost, 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, or 10⁹, unique molecular labelsequences. Barcodes with unique molecular label sequences can beattached to a given solid support (e.g., a bead).

For stochastic barcoding using a plurality of stochastic barcodes, theratio of the number of different molecular label sequences and thenumber of occurrence of any of the targets can be, or be about, 1:1,2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1,15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1,90:1, 100:1, or a number or a range between any two of these values. Atarget can be an mRNA species comprising mRNA molecules with identicalor nearly identical sequences. In some embodiments, the ratio of thenumber of different molecular label sequences and the number ofoccurrence of any of the targets is at least, or is at most, 1:1, 2:1,3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1,16:1, 17:1, 18:1, 19:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1,or 100:1.

A molecular label can be, or be about, 1, 2, 3, 4, 5, 10, 15, 20, 25,30, 35, 40, 45, 50, or a number or a range between any two of thesevalues, nucleotides in length. A molecular label can be at least, or beat most, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or300 nucleotides in length.

Target-Binding Region

A barcode can comprise one or more target binding regions, such ascapture probes. In some embodiments, a target-binding region canhybridize with a target of interest. In some embodiments, the targetbinding regions can comprise a nucleic acid sequence that hybridizesspecifically to a target (e.g., target nucleic acid, target molecule,e.g., a cellular nucleic acid to be analyzed), for example to a specificgene sequence. In some embodiments, a target binding region can comprisea nucleic acid sequence that can attach (e.g., hybridize) to a specificlocation of a specific target nucleic acid. In some embodiments, thetarget binding region can comprise a nucleic acid sequence that iscapable of specific hybridization to a restriction enzyme site overhang(e.g., an EcoRI sticky-end overhang). The barcode can then ligate to anynucleic acid molecule comprising a sequence complementary to therestriction site overhang.

In some embodiments, a target binding region can comprise a non-specifictarget nucleic acid sequence. A non-specific target nucleic acidsequence can refer to a sequence that can bind to multiple targetnucleic acids, independent of the specific sequence of the targetnucleic acid. For example, target binding region can comprise a randommultimer sequence, or an oligo(dT) sequence that hybridizes to thepoly(A) tail on mRNA molecules. A random multimer sequence can be, forexample, a random dimer, trimer, quatramer, pentamer, hexamer, septamer,octamer, nonamer, decamer, or higher multimer sequence of any length. Insome embodiments, the target binding region is the same for all barcodesattached to a given bead. In some embodiments, the target bindingregions for the plurality of barcodes attached to a given bead cancomprise two or more different target binding sequences. A targetbinding region can be, or be about, 5, 10, 15, 20, 25, 30, 35, 40, 45,50, or a number or a range between any two of these values, nucleotidesin length. A target binding region can be at most about 5, 10, 15, 20,25, 30, 35, 40, 45, 50 or more nucleotides in length.

In some embodiments, a target-binding region can comprise an oligo(dT)which can hybridize with mRNAs comprising polyadenylated ends. Atarget-binding region can be gene-specific. For example, atarget-binding region can be configured to hybridize to a specificregion of a target. A target-binding region can be, or be about, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26 27, 28, 29, 30, or a number or a range between any two ofthese values, nucleotides in length. A target-binding region can be atleast, or be at most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 27, 28, 29, or 30,nucleotides in length. A target-binding region can be about 5-30nucleotides in length. When a barcode comprises a gene-specifictarget-binding region, the barcode can be referred to herein as agene-specific barcode.

Orientation Property

A stochastic barcode (e.g., a stochastic barcode) can comprise one ormore orientation properties which can be used to orient (e.g., align)the barcodes. A barcode can comprise a moiety for isoelectric focusing.Different barcodes can comprise different isoelectric focusing points.When these barcodes are introduced to a sample, the sample can undergoisoelectric focusing in order to orient the barcodes into a known way.In this way, the orientation property can be used to develop a known mapof barcodes in a sample. Exemplary orientation properties can include,electrophoretic mobility (e.g., based on size of the barcode),isoelectric point, spin, conductivity, and/or self-assembly. Forexample, barcodes with an orientation property of self-assembly, canself-assemble into a specific orientation (e.g., nucleic acidnanostructure) upon activation.

Affinity Property

A barcode (e.g., a stochastic barcode) can comprise one or more affinityproperties. For example, a spatial label can comprise an affinityproperty. An affinity property can include a chemical and/or biologicalmoiety that can facilitate binding of the barcode to another entity(e.g., cell receptor). For example, an affinity property can comprise anantibody, for example, an antibody specific for a specific moiety (e.g.,receptor) on a sample. In some embodiments, the antibody can guide thebarcode to a specific cell type or molecule. Targets at and/or near thespecific cell type or molecule can be labeled (e.g., stochasticallylabeled). The affinity property can, in some embodiments, providespatial information in addition to the nucleotide sequence of thespatial label because the antibody can guide the barcode to a specificlocation. The antibody can be a therapeutic antibody, for example amonoclonal antibody or a polyclonal antibody. The antibody can behumanized or chimeric. The antibody can be a naked antibody or a fusionantibody.

The antibody can be a full-length (i.e., naturally occurring or formedby normal immunoglobulin gene fragment recombinatorial processes)immunoglobulin molecule (e.g., an IgG antibody) or an immunologicallyactive (i.e., specifically binding) portion of an immunoglobulinmolecule, like an antibody fragment.

The antibody fragment can be, for example, a portion of an antibody suchas F(ab′)2, Fab′, Fab, Fv, sFv and the like. In some embodiments, theantibody fragment can bind with the same antigen that is recognized bythe full-length antibody. The antibody fragment can include isolatedfragments consisting of the variable regions of antibodies, such as the“Fv” fragments consisting of the variable regions of the heavy and lightchains and recombinant single chain polypeptide molecules in which lightand heavy variable regions are connected by a peptide linker (“scFvproteins”). Exemplary antibodies can include, but are not limited to,antibodies for cancer cells, antibodies for viruses, antibodies thatbind to cell surface receptors (CD8, CD34, CD45), and therapeuticantibodies.

Universal Adaptor Primer

A barcode can comprise one or more universal adaptor primers. Forexample, a gene-specific barcode, such as a gene-specific stochasticbarcode, can comprise a universal adaptor primer. A universal adaptorprimer can refer to a nucleotide sequence that is universal across allbarcodes. A universal adaptor primer can be used for buildinggene-specific barcodes. A universal adaptor primer can be, or be about,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26 27, 28, 29, 30, or a number or a range betweenany two of these nucleotides in length. A universal adaptor primer canbe at least, or be at most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 27, 28, 29, or 30nucleotides in length. A universal adaptor primer can be from 5-30nucleotides in length.

Linker

When a barcode comprises more than one of a type of label (e.g., morethan one cell label or more than one barcode sequence, such as onemolecular label), the labels may be interspersed with a linker labelsequence. A linker label sequence can be at least about 5, 10, 15, 20,25, 30, 35, 40, 45, 50 or more nucleotides in length. A linker labelsequence can be at most about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 ormore nucleotides in length. In some instances, a linker label sequenceis 12 nucleotides in length. A linker label sequence can be used tofacilitate the synthesis of the barcode. The linker label can comprisean error-correcting (e.g., Hamming) code.

Solid Supports

Barcodes, such as stochastic barcodes, disclosed herein can, in someembodiments, be associated with a solid support. The solid support canbe, for example, a synthetic particle. In some embodiments, some or allof the barcode sequences, such as molecular labels for stochasticbarcodes (e.g., the first barcode sequences) of a plurality of barcodes(e.g., the first plurality of barcodes) on a solid support differ by atleast one nucleotide. The cell labels of the barcodes on the same solidsupport can be the same. The cell labels of the barcodes on differentsolid supports can differ by at least one nucleotide. For example, firstcell labels of a first plurality of barcodes on a first solid supportcan have the same sequence, and second cell labels of a second pluralityof barcodes on a second solid support can have the same sequence. Thefirst cell labels of the first plurality of barcodes on the first solidsupport and the second cell labels of the second plurality of barcodeson the second solid support can differ by at least one nucleotide. Acell label can be, for example, about 5-20 nucleotides long. A barcodesequence can be, for example, about 5-20 nucleotides long. The syntheticparticle can be, for example, a bead.

The bead can be, for example, a silica gel bead, a controlled pore glassbead, a magnetic bead, a Dynabead, a Sephadex/Sepharose bead, acellulose bead, a polystyrene bead, or any combination thereof. The beadcan comprise a material such as polydimethylsiloxane (PDMS),polystyrene, glass, polypropylene, agarose, gelatin, hydrogel,paramagnetic, ceramic, plastic, glass, methylstyrene, acrylic polymer,titanium, latex, Sepharose, cellulose, nylon, silicone, or anycombination thereof.

In some embodiments, the bead can be a polymeric bead, for example adeformable bead or a gel bead, functionalized with barcodes orstochastic barcodes (such as gel beads from 10× Genomics (San Francisco,Calif.). In some implementation, a gel bead can comprise a polymer-basedgels. Gel beads can be generated, for example, by encapsulating one ormore polymeric precursors into droplets. Upon exposure of the polymericprecursors to an accelerator (e.g., tetramethylethylenediamine (TEMED)),a gel bead may be generated.

In some embodiments, the particle can be degradable. For example, thepolymeric bead can dissolve, melt, or degrade, for example, under adesired condition. The desired condition can include an environmentalcondition. The desired condition may result in the polymeric beaddissolving, melting, or degrading in a controlled manner. A gel bead maydissolve, melt, or degrade due to a chemical stimulus, a physicalstimulus, a biological stimulus, a thermal stimulus, a magneticstimulus, an electric stimulus, a light stimulus, or any combinationthereof.

Analytes and/or reagents, such as oligonucleotide barcodes, for example,may be coupled/immobilized to the interior surface of a gel bead (e.g.,the interior accessible via diffusion of an oligonucleotide barcodeand/or materials used to generate an oligonucleotide barcode) and/or theouter surface of a gel bead or any other microcapsule described herein.Coupling/immobilization may be via any form of chemical bonding (e.g.,covalent bond, ionic bond) or physical phenomena (e.g., Van der Waalsforces, dipole-dipole interactions, etc.). In some embodiments,coupling/immobilization of a reagent to a gel bead or any othermicrocapsule described herein may be reversible, such as, for example,via a labile moiety (e.g., via a chemical cross-linker, includingchemical cross-linkers described herein). Upon application of astimulus, the labile moiety may be cleaved and the immobilized reagentset free. In some embodiments, the labile moiety is a disulfide bond.For example, in the case where an oligonucleotide barcode is immobilizedto a gel bead via a disulfide bond, exposure of the disulfide bond to areducing agent can cleave the disulfide bond and free theoligonucleotide barcode from the bead. The labile moiety may be includedas part of a gel bead or microcapsule, as part of a chemical linker thatlinks a reagent or analyte to a gel bead or microcapsule, and/or as partof a reagent or analyte. In some embodiments, at least one barcode ofthe plurality of barcodes can be immobilized on the particle, partiallyimmobilized on the particle, enclosed in the particle, partiallyenclosed in the particle, or any combination thereof.

In some embodiments, a gel bead can comprise a wide range of differentpolymers including but not limited to: polymers, heat sensitivepolymers, photosensitive polymers, magnetic polymers, pH sensitivepolymers, salt-sensitive polymers, chemically sensitive polymers,polyelectrolytes, polysaccharides, peptides, proteins, and/or plastics.Polymers may include but are not limited to materials such aspoly(N-isopropylacrylamide) (PNIPAAm), poly(styrene sulfonate) (PSS),poly(allyl amine) (PAAm), poly(acrylic acid) (PAA), poly(ethylene imine)(PEI), poly(diallyldimethyl-ammonium chloride) (PDADMAC), poly(pyrolle)(PPy), poly(vinylpyrrolidone) (PVPON), poly(vinyl pyridine) (PVP),poly(methacrylic acid) (PMAA), poly(methyl methacrylate) (PMMA),polystyrene (PS), poly(tetrahydrofuran) (PTHF), poly(phthaladehyde)(PTHF), poly(hexyl viologen) (PHV), poly(L-lysine) (PLL),poly(L-arginine) (PARG), poly(lactic-co-glycolic acid) (PLGA).

Numerous chemical stimuli can be used to trigger the disruption,dissolution, or degradation of the beads. Examples of these chemicalchanges may include, but are not limited to pH-mediated changes to thebead wall, disintegration of the bead wall via chemical cleavage ofcrosslink bonds, triggered depolymerization of the bead wall, and beadwall switching reactions. Bulk changes may also be used to triggerdisruption of the beads.

Bulk or physical changes to the microcapsule through various stimulialso offer many advantages in designing capsules to release reagents.Bulk or physical changes occur on a macroscopic scale, in which beadrupture is the result of mechano-physical forces induced by a stimulus.These processes may include, but are not limited to pressure inducedrupture, bead wall melting, or changes in the porosity of the bead wall.

Biological stimuli may also be used to trigger disruption, dissolution,or degradation of beads. Generally, biological triggers resemblechemical triggers, but many examples use biomolecules, or moleculescommonly found in living systems such as enzymes, peptides, saccharides,fatty acids, nucleic acids and the like. For example, beads may comprisepolymers with peptide cross-links that are sensitive to cleavage byspecific proteases. More specifically, one example may comprise amicrocapsule comprising GFLGK peptide cross links. Upon addition of abiological trigger such as the protease Cathepsin B, the peptide crosslinks of the shell well are cleaved and the contents of the beads arereleased. In other cases, the proteases may be heat-activated. Inanother example, beads comprise a shell wall comprising cellulose.Addition of the hydrolytic enzyme chitosan serves as biologic triggerfor cleavage of cellulosic bonds, depolymerization of the shell wall,and release of its inner contents.

The beads may also be induced to release their contents upon theapplication of a thermal stimulus. A change in temperature can cause avariety changes to the beads. A change in heat may cause melting of abead such that the bead wall disintegrates. In other cases, the heat mayincrease the internal pressure of the inner components of the bead suchthat the bead ruptures or explodes. In still other cases, the heat maytransform the bead into a shrunken dehydrated state. The heat may alsoact upon heat-sensitive polymers within the wall of a bead to causedisruption of the bead.

Inclusion of magnetic nanoparticles to the bead wall of microcapsulesmay allow triggered rupture of the beads as well as guide the beads inan array. A device of this disclosure may comprise magnetic beads foreither purpose. In one example, incorporation of Fe₃O₄ nanoparticlesinto polyelectrolyte containing beads triggers rupture in the presenceof an oscillating magnetic field stimulus.

A bead may also be disrupted, dissolved, or degraded as the result ofelectrical stimulation. Similar to magnetic particles described in theprevious section, electrically sensitive beads can allow for bothtriggered rupture of the beads as well as other functions such asalignment in an electric field, electrical conductivity or redoxreactions. In one example, beads containing electrically sensitivematerial are aligned in an electric field such that release of innerreagents can be controlled. In other examples, electrical fields mayinduce redox reactions within the bead wall itself that may increaseporosity.

A light stimulus may also be used to disrupt the beads. Numerous lighttriggers are possible and may include systems that use various moleculessuch as nanoparticles and chromophores capable of absorbing photons ofspecific ranges of wavelengths. For example, metal oxide coatings can beused as capsule triggers. UV irradiation of polyelectrolyte capsulescoated with SiO₂ may result in disintegration of the bead wall. In yetanother example, photo switchable materials such as azobenzene groupsmay be incorporated in the bead wall. Upon the application of UV orvisible light, chemicals such as these undergo a reversible cis-to-transisomerization upon absorption of photons. In this aspect, incorporationof photon switches result in a bead wall that may disintegrate or becomemore porous upon the application of a light trigger.

For example, in a non-limiting example of barcoding (e.g., stochasticbarcoding) illustrated in FIG. 2, after introducing cells such as singlecells onto a plurality of microwells of a microwell array at block 208,beads can be introduced onto the plurality of microwells of themicrowell array at block 212. Each microwell can comprise one bead. Thebeads can comprise a plurality of barcodes. A barcode can comprise a 5′amine region attached to a bead. The barcode can comprise a universallabel, a barcode sequence (e.g., a molecular label), a target-bindingregion, or any combination thereof.

The barcodes disclosed herein can be associated with (e.g., attached to)a solid support (e.g., a bead). The barcodes associated with a solidsupport can each comprise a barcode sequence selected from a groupcomprising at least 100 or 1000 barcode sequences with unique sequences.In some embodiments, different barcodes associated with a solid supportcan comprise barcode with different sequences. In some embodiments, apercentage of barcodes associated with a solid support comprises thesame cell label. For example, the percentage can be, or be about 60%,70%, 80%, 85%, 90%, 95%, 97%, 99%, 100%, or a number or a range betweenany two of these values. As another example, the percentage can be atleast, or be at most 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or 100%. Insome embodiments, barcodes associated with a solid support can have thesame cell label. The barcodes associated with different solid supportscan have different cell labels selected from a group comprising at least100 or 1000 cell labels with unique sequences.

The barcodes disclosed herein can be associated to (e.g., attached to) asolid support (e.g., a bead). In some embodiments, barcoding theplurality of targets in the sample can be performed with a solid supportincluding a plurality of synthetic particles associated with theplurality of barcodes. In some embodiments, the solid support caninclude a plurality of synthetic particles associated with the pluralityof barcodes. The spatial labels of the plurality of barcodes ondifferent solid supports can differ by at least one nucleotide. Thesolid support can, for example, include the plurality of barcodes in twodimensions or three dimensions. The synthetic particles can be beads.The beads can be silica gel beads, controlled pore glass beads, magneticbeads, Dynabeads, Sephadex/Sepharose beads, cellulose beads, polystyrenebeads, or any combination thereof. The solid support can include apolymer, a matrix, a hydrogel, a needle array device, an antibody, orany combination thereof. In some embodiments, the solid supports can befree floating. In some embodiments, the solid supports can be embeddedin a semi-solid or solid array. The barcodes may not be associated withsolid supports. The barcodes can be individual nucleotides. The barcodescan be associated with a substrate.

As used herein, the terms “tethered,” “attached,” and “immobilized,” areused interchangeably, and can refer to covalent or non-covalent meansfor attaching barcodes to a solid support. Any of a variety of differentsolid supports can be used as solid supports for attachingpre-synthesized barcodes or for in situ solid-phase synthesis ofbarcode.

In some embodiments, the solid support is a bead. The bead can compriseone or more types of solid, porous, or hollow sphere, ball, bearing,cylinder, or other similar configuration which a nucleic acid can beimmobilized (e.g., covalently or non-covalently). The bead can be, forexample, composed of plastic, ceramic, metal, polymeric material, or anycombination thereof. A bead can be, or comprise, a discrete particlethat is spherical (e.g., microspheres) or have a non-spherical orirregular shape, such as cubic, cuboid, pyramidal, cylindrical, conical,oblong, or disc-shaped, and the like. In some embodiments, a bead can benon-spherical in shape.

Beads can comprise a variety of materials including, but not limited to,paramagnetic materials (e.g., magnesium, molybdenum, lithium, andtantalum), superparamagnetic materials (e.g., ferrite (Fe₃O₄; magnetite)nanoparticles), ferromagnetic materials (e.g., iron, nickel, cobalt,some alloys thereof, and some rare earth metal compounds), ceramic,plastic, glass, polystyrene, silica, methylstyrene, acrylic polymers,titanium, latex, Sepharose, agarose, hydrogel, polymer, cellulose,nylon, or any combination thereof.

In some embodiments, the bead (e.g., the bead to which the labels areattached) is a hydrogel bead. In some embodiments, the bead compriseshydrogel.

Some embodiments disclosed herein include one or more particles (forexample, beads). Each of the particles can comprise a plurality ofoligonucleotides (e.g., barcodes). Each of the plurality ofoligonucleotides can comprise a barcode sequence (e.g., a molecularlabel sequence), a cell label, and a target-binding region (e.g., anoligo(dT) sequence, a gene-specific sequence, a random multimer, or acombination thereof). The cell label sequence of each of the pluralityof oligonucleotides can be the same. The cell label sequences ofoligonucleotides on different particles can be different such that theoligonucleotides on different particles can be identified. The number ofdifferent cell label sequences can be different in differentimplementations. In some embodiments, the number of cell label sequencescan be, or be about 10, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000,30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 10⁶, 10⁷, 10⁸,10⁹, a number or a range between any two of these values, or more. Insome embodiments, the number of cell label sequences can be at least, orbe at most 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000,3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000,50000, 60000, 70000, 80000, 90000, 100000, 10⁶, 10⁷, 10⁸, or 10⁹. Insome embodiments, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, or more of the plurality of the particles include oligonucleotideswith the same cell sequence. In some embodiment, the plurality ofparticles that include oligonucleotides with the same cell sequence canbe at most 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or more. In some embodiments, none ofthe plurality of the particles has the same cell label sequence.

The plurality of oligonucleotides on each particle can comprisedifferent barcode sequences (e.g., molecular labels). In someembodiments, the number of barcode sequences can be, or be about 10,100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000,5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000,70000, 80000, 90000, 100000, 10⁶, 10⁷, 10⁸, 10⁹, or a number or a rangebetween any two of these values. In some embodiments, the number ofbarcode sequences can be at least, or be at most 10, 100, 200, 300, 400,500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000,9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000,100000, 10⁶, 10⁷, 10⁸, or 10⁹. For example, at least 100 of theplurality of oligonucleotides comprise different barcode sequences. Asanother example, in a single particle, at least 100, 500, 1000, 5000,10000, 15000, 20000, 50000, a number or a range between any two of thesevalues, or more of the plurality of oligonucleotides comprise differentbarcode sequences. Some embodiments provide a plurality of the particlescomprising barcodes. In some embodiments, the ratio of an occurrence (ora copy or a number) of a target to be labeled and the different barcodesequences can be at least 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9,1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:30,1:40, 1:50, 1:60, 1:70, 1:80, 1:90, or more. In some embodiments, eachof the plurality of oligonucleotides further comprises a sample label, auniversal label, or both. The particle can be, for example, ananoparticle or microparticle.

The size of the beads can vary. For example, the diameter of the beadcan range from 0.1 micrometer to 50 micrometers. In some embodiments,the diameter of the bead can be, or be about, 0.1, 0.5, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 20, 30, 40, 50 micrometers, or a number or a rangebetween any two of these values.

The diameter of the bead can be related to the diameter of the wells ofthe substrate. In some embodiments, the diameter of the bead can be, orbe about, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or a numberor a range between any two of these values, longer or shorter than thediameter of the well. The diameter of the beads can be related to thediameter of a cell (e.g., a single cell entrapped by a well of thesubstrate). In some embodiments, the diameter of the bead can be atleast, or be at most, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or100% longer or shorter than the diameter of the well. The diameter ofthe beads can be related to the diameter of a cell (e.g., a single cellentrapped by a well of the substrate). In some embodiments, the diameterof the bead can be, or be about, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100%, 150%, 200%, 250%, 300%, or a number or a range between anytwo of these values, longer or shorter than the diameter of the cell. Insome embodiments, the diameter of the beads can be at least, or be atmost, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%,250%, or 300% longer or shorter than the diameter of the cell.

A bead can be attached to and/or embedded in a substrate. A bead can beattached to and/or embedded in a gel, hydrogel, polymer and/or matrix.The spatial position of a bead within a substrate (e.g., gel, matrix,scaffold, or polymer) can be identified using the spatial label presenton the barcode on the bead which can serve as a location address.

Examples of beads can include, but are not limited to, streptavidinbeads, agarose beads, magnetic beads, Dynabeads®, MACS® microbeads,antibody conjugated beads (e.g., anti-immunoglobulin microbeads),protein A conjugated beads, protein G conjugated beads, protein A/Gconjugated beads, protein L conjugated beads, oligo(dT) conjugatedbeads, silica beads, silica-like beads, anti-biotin microbeads,anti-fluorochrome microbeads, and BcMag™ Carboxyl-Terminated MagneticBeads.

A bead can be associated with (e.g., impregnated with) quantum dots orfluorescent dyes to make it fluorescent in one fluorescence opticalchannel or multiple optical channels. A bead can be associated with ironoxide or chromium oxide to make it paramagnetic or ferromagnetic. Beadscan be identifiable. For example, a bead can be imaged using a camera. Abead can have a detectable code associated with the bead. For example, abead can comprise a barcode. A bead can change size, for example, due toswelling in an organic or inorganic solution. A bead can be hydrophobic.A bead can be hydrophilic. A bead can be biocompatible.

A solid support (e.g., a bead) can be visualized. The solid support cancomprise a visualizing tag (e.g., fluorescent dye). A solid support(e.g., a bead) can be etched with an identifier (e.g., a number). Theidentifier can be visualized through imaging the beads.

A solid support can comprise an insoluble, semi-soluble, or insolublematerial. A solid support can be referred to as “functionalized” when itincludes a linker, a scaffold, a building block, or other reactivemoiety attached thereto, whereas a solid support may be“nonfunctionalized” when it lacks such a reactive moiety attachedthereto. The solid support can be employed free in solution, such as ina microtiter well format; in a flow-through format, such as in a column;or in a dipstick.

The solid support can comprise a membrane, paper, plastic, coatedsurface, flat surface, glass, slide, chip, or any combination thereof. Asolid support can take the form of resins, gels, microspheres, or othergeometric configurations. A solid support can comprise silica chips,microparticles, nanoparticles, plates, arrays, capillaries, flatsupports such as glass fiber filters, glass surfaces, metal surfaces(steel, gold silver, aluminum, silicon and copper), glass supports,plastic supports, silicon supports, chips, filters, membranes, microwellplates, slides, plastic materials including multiwell plates ormembranes (e.g., formed of polyethylene, polypropylene, polyamide,polyvinylidenedifluoride), and/or wafers, combs, pins or needles (e.g.,arrays of pins suitable for combinatorial synthesis or analysis) orbeads in an array of pits or nanoliter wells of flat surfaces such aswafers (e.g., silicon wafers), wafers with pits with or without filterbottoms.

The solid support can comprise a polymer matrix (e.g., gel, hydrogel).The polymer matrix may be able to permeate intracellular space (e.g.,around organelles). The polymer matrix may able to be pumped throughoutthe circulatory system.

Substrates and Microwell Array

As used herein, a substrate can refer to a type of solid support. Asubstrate can refer to a solid support that can comprise barcodes orstochastic barcodes of the disclosure. A substrate can, for example,comprise a plurality of microwells. For example, a substrate can be awell array comprising two or more microwells. In some embodiments, amicrowell can comprise a small reaction chamber of defined volume. Insome embodiments, a microwell can entrap one or more cells. In someembodiments, a microwell can entrap only one cell. In some embodiments,a microwell can entrap one or more solid supports. In some embodiments,a microwell can entrap only one solid support. In some embodiments, amicrowell entraps a single cell and a single solid support (e.g., abead). A microwell can comprise barcode reagents of the disclosure.

Methods of Barcoding

The disclosure provides for methods for estimating the number ofdistinct targets at distinct locations in a physical sample (e.g.,tissue, organ, tumor, cell). The methods can comprise placing barcodes(e.g., stochastic barcodes) in close proximity with the sample, lysingthe sample, associating distinct targets with the barcodes, amplifyingthe targets and/or digitally counting the targets. The method canfurther comprise analyzing and/or visualizing the information obtainedfrom the spatial labels on the barcodes. In some embodiments, a methodcomprises visualizing the plurality of targets in the sample. Mappingthe plurality of targets onto the map of the sample can includegenerating a two dimensional map or a three dimensional map of thesample. The two dimensional map and the three dimensional map can begenerated prior to or after barcoding (e.g., stochastically barcoding)the plurality of targets in the sample. Visualizing the plurality oftargets in the sample can include mapping the plurality of targets ontoa map of the sample. Mapping the plurality of targets onto the map ofthe sample can include generating a two dimensional map or a threedimensional map of the sample. The two dimensional map and the threedimensional map can be generated prior to or after barcoding theplurality of targets in the sample. In some embodiments, the twodimensional map and the three dimensional map can be generated before orafter lysing the sample. Lysing the sample before or after generatingthe two dimensional map or the three dimensional map can include heatingthe sample, contacting the sample with a detergent, changing the pH ofthe sample, or any combination thereof.

In some embodiments, barcoding the plurality of targets compriseshybridizing a plurality of barcodes with a plurality of targets tocreate barcoded targets (e.g., stochastically barcoded targets).Barcoding the plurality of targets can comprise generating an indexedlibrary of the barcoded targets. Generating an indexed library of thebarcoded targets can be performed with a solid support comprising theplurality of barcodes (e.g., stochastic barcodes).

Contacting a Sample and a Barcode

The disclosure provides for methods for contacting a sample (e.g.,cells) to a substrate of the disclosure. A sample comprising, forexample, a cell, organ, or tissue thin section, can be contacted tobarcodes (e.g., stochastic barcodes). The cells can be contacted, forexample, by gravity flow wherein the cells can settle and create amonolayer. The sample can be a tissue thin section. The thin section canbe placed on the substrate. The sample can be one-dimensional (e.g.,forms a planar surface). The sample (e.g., cells) can be spread acrossthe substrate, for example, by growing/culturing the cells on thesubstrate.

When barcodes are in close proximity to targets, the targets canhybridize to the barcode. The barcodes can be contacted at anon-depletable ratio such that each distinct target can associate with adistinct barcode of the disclosure. To ensure efficient associationbetween the target and the barcode, the targets can be cross-linked tobarcode.

Cell Lysis

Following the distribution of cells and barcodes, the cells can be lysedto liberate the target molecules. Cell lysis can be accomplished by anyof a variety of means, for example, by chemical or biochemical means, byosmotic shock, or by means of thermal lysis, mechanical lysis, oroptical lysis. Cells can be lysed by addition of a cell lysis buffercomprising a detergent (e.g., SDS, Li dodecyl sulfate, Triton X-100,Tween-20, or NP-40), an organic solvent (e.g., methanol or acetone), ordigestive enzymes (e.g., proteinase K, pepsin, or trypsin), or anycombination thereof. To increase the association of a target and abarcode, the rate of the diffusion of the target molecules can bealtered by for example, reducing the temperature and/or increasing theviscosity of the lysate.

In some embodiments, the sample can be lysed using a filter paper. Thefilter paper can be soaked with a lysis buffer on top of the filterpaper. The filter paper can be applied to the sample with pressure whichcan facilitate lysis of the sample and hybridization of the targets ofthe sample to the substrate.

In some embodiments, lysis can be performed by mechanical lysis, heatlysis, optical lysis, and/or chemical lysis. Chemical lysis can includethe use of digestive enzymes such as proteinase K, pepsin, and trypsin.Lysis can be performed by the addition of a lysis buffer to thesubstrate. A lysis buffer can comprise Tris HCl. A lysis buffer cancomprise at least about 0.01, 0.05, 0.1, 0.5, or 1 M or more Tris HCl. Alysis buffer can comprise at most about 0.01, 0.05, 0.1, 0.5, or 1 M ormore Tris HCL. A lysis buffer can comprise about 0.1 M Tris HCl. The pHof the lysis buffer can be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more. The pH of the lysis buffer can be at most about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, or more. In some embodiments, the pH of the lysis bufferis about 7.5. The lysis buffer can comprise a salt (e.g., LiCl). Theconcentration of salt in the lysis buffer can be at least about 0.1,0.5, or 1 M or more. The concentration of salt in the lysis buffer canbe at most about 0.1, 0.5, or 1 M or more. In some embodiments, theconcentration of salt in the lysis buffer is about 0.5M. The lysisbuffer can comprise a detergent (e.g., SDS, Li dodecyl sulfate, tritonX, tween, NP-40). The concentration of the detergent in the lysis buffercan be at least about 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%,0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, or 7%, or more. The concentration ofthe detergent in the lysis buffer can be at most about 0.0001%, 0.0005%,0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, or 7%,or more. In some embodiments, the concentration of the detergent in thelysis buffer is about 1% Li dodecyl sulfate. The time used in the methodfor lysis can be dependent on the amount of detergent used. In someembodiments, the more detergent used, the less time needed for lysis.The lysis buffer can comprise a chelating agent (e.g., EDTA, EGTA). Theconcentration of a chelating agent in the lysis buffer can be at leastabout 1, 5, 10, 15, 20, 25, or 30 mM or more. The concentration of achelating agent in the lysis buffer can be at most about 1, 5, 10, 15,20, 25, or 30 mM or more. In some embodiments, the concentration ofchelating agent in the lysis buffer is about 10 mM. The lysis buffer cancomprise a reducing reagent (e.g., beta-mercaptoethanol, DTT). Theconcentration of the reducing reagent in the lysis buffer can be atleast about 1, 5, 10, 15, or 20 mM or more. The concentration of thereducing reagent in the lysis buffer can be at most about 1, 5, 10, 15,or 20 mM or more. In some embodiments, the concentration of reducingreagent in the lysis buffer is about 5 mM. In some embodiments, a lysisbuffer can comprise about 0.1M TrisHCl, about pH 7.5, about 0.5M LiCl,about 1% lithium dodecyl sulfate, about 10 mM EDTA, and about 5 mM DTT.

Lysis can be performed at a temperature of about 4, 10, 15, 20, 25, or30° C. Lysis can be performed for about 1, 5, 10, 15, or 20 or moreminutes. A lysed cell can comprise at least about 100000, 200000,300000, 400000, 500000, 600000, or 700000 or more target nucleic acidmolecules. A lysed cell can comprise at most about 100000, 200000,300000, 400000, 500000, 600000, or 700000 or more target nucleic acidmolecules.

Attachment of Barcodes to Target Nucleic Acid Molecules

Following lysis of the cells and release of nucleic acid moleculestherefrom, the nucleic acid molecules can randomly associate with thebarcodes of the co-localized solid support. Association can comprisehybridization of a barcode's target recognition region to acomplementary portion of the target nucleic acid molecule (e.g.,oligo(dT) of the barcode can interact with a poly(A) tail of a target).The assay conditions used for hybridization (e.g., buffer pH, ionicstrength, temperature, etc.) can be chosen to promote formation ofspecific, stable hybrids. In some embodiments, the nucleic acidmolecules released from the lysed cells can associate with the pluralityof probes on the substrate (e.g., hybridize with the probes on thesubstrate). When the probes comprise oligo(dT), mRNA molecules canhybridize to the probes and be reverse transcribed. The oligo(dT)portion of the oligonucleotide can act as a primer for first strandsynthesis of the cDNA molecule. For example, in a non-limiting exampleof barcoding illustrated in FIG. 2, at block 216, mRNA molecules canhybridize to barcodes on beads. For example, single-stranded nucleotidefragments can hybridize to the target-binding regions of barcodes.

Attachment can further comprise ligation of a barcode's targetrecognition region and a portion of the target nucleic acid molecule.For example, the target binding region can comprise a nucleic acidsequence that can be capable of specific hybridization to a restrictionsite overhang (e.g., an EcoRI sticky-end overhang). The assay procedurecan further comprise treating the target nucleic acids with arestriction enzyme (e.g., EcoRI) to create a restriction site overhang.The barcode can then be ligated to any nucleic acid molecule comprisinga sequence complementary to the restriction site overhang. A ligase(e.g., T4 DNA ligase) can be used to join the two fragments.

For example, in a non-limiting example of barcoding illustrated in FIG.2, at block 220, the labeled targets from a plurality of cells (or aplurality of samples) (e.g., target-barcode molecules) can besubsequently pooled, for example, into a tube. The labeled targets canbe pooled by, for example, retrieving the barcodes and/or the beads towhich the target-barcode molecules are attached.

The retrieval of solid support-based collections of attachedtarget-barcode molecules can be implemented by use of magnetic beads andan externally-applied magnetic field. Once the target-barcode moleculeshave been pooled, all further processing can proceed in a singlereaction vessel. Further processing can include, for example, reversetranscription reactions, amplification reactions, cleavage reactions,dissociation reactions, and/or nucleic acid extension reactions. Furtherprocessing reactions can be performed within the microwells, that is,without first pooling the labeled target nucleic acid molecules from aplurality of cells.

Reverse Transcription

The disclosure provides for a method to create a target-barcodeconjugate using reverse transcription (e.g., at block 224 of FIG. 2).The target-barcode conjugate can comprise the barcode and acomplementary sequence of all or a portion of the target nucleic acid(i.e., a barcoded cDNA molecule, such as a stochastically barcoded cDNAmolecule). Reverse transcription of the associated RNA molecule canoccur by the addition of a reverse transcription primer along with thereverse transcriptase. The reverse transcription primer can be anoligo(dT) primer, a random hexanucleotide primer, or a target-specificoligonucleotide primer. Oligo(dT) primers can be, or can be about, 12-18nucleotides in length and bind to the endogenous poly(A) tail at the 3′end of mammalian mRNA. Random hexanucleotide primers can bind to mRNA ata variety of complementary sites. Target-specific oligonucleotideprimers typically selectively prime the mRNA of interest.

In some embodiments, reverse transcription of the labeled-RNA moleculecan occur by the addition of a reverse transcription primer. In someembodiments, the reverse transcription primer is an oligo(dT) primer,random hexanucleotide primer, or a target-specific oligonucleotideprimer. Generally, oligo(dT) primers are 12-18 nucleotides in length andbind to the endogenous poly(A) tail at the 3′ end of mammalian mRNA.Random hexanucleotide primers can bind to mRNA at a variety ofcomplementary sites. Target-specific oligonucleotide primers typicallyselectively prime the mRNA of interest.

Reverse transcription can occur repeatedly to produce multiplelabeled-cDNA molecules. The methods disclosed herein can compriseconducting at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 reverse transcription reactions. The methodcan comprise conducting at least about 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, or 100 reverse transcription reactions.

Amplification

One or more nucleic acid amplification reactions (e.g., at block 228 ofFIG. 2) can be performed to create multiple copies of the labeled targetnucleic acid molecules. Amplification can be performed in a multiplexedmanner, wherein multiple target nucleic acid sequences are amplifiedsimultaneously. The amplification reaction can be used to add sequencingadaptors to the nucleic acid molecules. The amplification reactions cancomprise amplifying at least a portion of a sample label, if present.The amplification reactions can comprise amplifying at least a portionof the cellular label and/or barcode sequence (e.g., a molecular label).The amplification reactions can comprise amplifying at least a portionof a sample tag, a cell label, a spatial label, a barcode sequence(e.g., a molecular label), a target nucleic acid, or a combinationthereof. The amplification reactions can comprise amplifying 0.5%, 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 100%, or a rangeor a number between any two of these values, of the plurality of nucleicacids. The method can further comprise conducting one or more cDNAsynthesis reactions to produce one or more cDNA copies of target-barcodemolecules comprising a sample label, a cell label, a spatial label,and/or a barcode sequence (e.g., a molecular label).

In some embodiments, amplification can be performed using a polymerasechain reaction (PCR). As used herein, PCR can refer to a reaction forthe in vitro amplification of specific DNA sequences by the simultaneousprimer extension of complementary strands of DNA. As used herein, PCRcan encompass derivative forms of the reaction, including but notlimited to, RT-PCR, real-time PCR, nested PCR, quantitative PCR,multiplexed PCR, digital PCR, and assembly PCR.

Amplification of the labeled nucleic acids can comprise non-PCR basedmethods. Examples of non-PCR based methods include, but are not limitedto, multiple displacement amplification (MDA), transcription-mediatedamplification (TMA), nucleic acid sequence-based amplification (NASBA),strand displacement amplification (SDA), real-time SDA, rolling circleamplification, or circle-to-circle amplification. Other non-PCR-basedamplification methods include multiple cycles of DNA-dependent RNApolymerase-driven RNA transcription amplification or RNA-directed DNAsynthesis and transcription to amplify DNA or RNA targets, a ligasechain reaction (LCR), and a Qβ replicase (Qβ) method, use of palindromicprobes, strand displacement amplification, oligonucleotide-drivenamplification using a restriction endonuclease, an amplification methodin which a primer is hybridized to a nucleic acid sequence and theresulting duplex is cleaved prior to the extension reaction andamplification, strand displacement amplification using a nucleic acidpolymerase lacking 5′ exonuclease activity, rolling circleamplification, and ramification extension amplification (RAM). In someembodiments, the amplification does not produce circularizedtranscripts.

In some embodiments, the methods disclosed herein further compriseconducting a polymerase chain reaction on the labeled nucleic acid(e.g., labeled-RNA, labeled-DNA, labeled-cDNA) to produce a labeledamplicon (e.g., a stochastically labeled amplicon). The labeled ampliconcan be double-stranded molecule. The double-stranded molecule cancomprise a double-stranded RNA molecule, a double-stranded DNA molecule,or a RNA molecule hybridized to a DNA molecule. One or both of thestrands of the double-stranded molecule can comprise a sample label, aspatial label, a cell label, and/or a barcode sequence (e.g., amolecular label). The labeled amplicon can be a single-strandedmolecule. The single-stranded molecule can comprise DNA, RNA, or acombination thereof. The nucleic acids of the disclosure can comprisesynthetic or altered nucleic acids.

Amplification can comprise use of one or more non-natural nucleotides.Non-natural nucleotides can comprise photolabile or triggerablenucleotides. Examples of non-natural nucleotides can include, but arenot limited to, peptide nucleic acid (PNA), morpholine and lockednucleic acid (LNA), as well as glycol nucleic acid (GNA) and threosenucleic acid (TNA). Non-natural nucleotides can be added to one or morecycles of an amplification reaction. The addition of the non-naturalnucleotides can be used to identify products as specific cycles or timepoints in the amplification reaction.

Conducting the one or more amplification reactions can comprise the useof one or more primers. The one or more primers can comprise, forexample, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or morenucleotides. The one or more primers can comprise at least 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more nucleotides. The one ormore primers can comprise less than 12-15 nucleotides. The one or moreprimers can anneal to at least a portion of the plurality of labeledtargets (e.g., stochastically labeled targets). The one or more primerscan anneal to the 3′ end or 5′ end of the plurality of labeled targets.The one or more primers can anneal to an internal region of theplurality of labeled targets. The internal region can be at least about50, 100, 150, 200, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310,320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450,460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590,600, 650, 700, 750, 800, 850, 900 or 1000 nucleotides from the 3′ endsthe plurality of labeled targets. The one or more primers can comprise afixed panel of primers. The one or more primers can comprise at leastone or more custom primers. The one or more primers can comprise atleast one or more control primers. The one or more primers can compriseat least one or more gene-specific primers.

The one or more primers can comprise a universal primer. The universalprimer can anneal to a universal primer binding site. The one or morecustom primers can anneal to a first sample label, a second samplelabel, a spatial label, a cell label, a barcode sequence (e.g., amolecular label), a target, or any combination thereof. The one or moreprimers can comprise a universal primer and a custom primer. The customprimer can be designed to amplify one or more targets. The targets cancomprise a subset of the total nucleic acids in one or more samples. Thetargets can comprise a subset of the total labeled targets in one ormore samples. The one or more primers can comprise at least 96 or morecustom primers. The one or more primers can comprise at least 960 ormore custom primers. The one or more primers can comprise at least 9600or more custom primers. The one or more custom primers can anneal to twoor more different labeled nucleic acids. The two or more differentlabeled nucleic acids can correspond to one or more genes.

Any amplification scheme can be used in the methods of the presentdisclosure. For example, in one scheme, the first round PCR can amplifymolecules attached to the bead using a gene specific primer and a primeragainst the universal Illumina sequencing primer 1 sequence. The secondround of PCR can amplify the first PCR products using a nested genespecific primer flanked by Illumina sequencing primer 2 sequence, and aprimer against the universal Illumina sequencing primer 1 sequence. Thethird round of PCR adds P5 and P7 and sample index to turn PCR productsinto an Illumina sequencing library. Sequencing using 150 bp×2sequencing can reveal the cell label and barcode sequence (e.g.,molecular label) on read 1, the gene on read 2, and the sample index onindex 1 read.

In some embodiments, nucleic acids can be removed from the substrateusing chemical cleavage. For example, a chemical group or a modifiedbase present in a nucleic acid can be used to facilitate its removalfrom a solid support. For example, an enzyme can be used to remove anucleic acid from a substrate. For example, a nucleic acid can beremoved from a substrate through a restriction endonuclease digestion.For example, treatment of a nucleic acid containing a dUTP or ddUTP withuracil-d-glycosylase (UDG) can be used to remove a nucleic acid from asubstrate. For example, a nucleic acid can be removed from a substrateusing an enzyme that performs nucleotide excision, such as a baseexcision repair enzyme, such as an apurinic/apyrimidinic (AP)endonuclease. In some embodiments, a nucleic acid can be removed from asubstrate using a photocleavable group and light. In some embodiments, acleavable linker can be used to remove a nucleic acid from thesubstrate. For example, the cleavable linker can comprise at least oneof biotin/avidin, biotin/streptavidin, biotin/neutravidin, Ig-protein A,a photo-labile linker, acid or base labile linker group, or an aptamer.

When the probes are gene-specific, the molecules can hybridize to theprobes and be reverse transcribed and/or amplified. In some embodiments,after the nucleic acid has been synthesized (e.g., reverse transcribed),it can be amplified. Amplification can be performed in a multiplexmanner, wherein multiple target nucleic acid sequences are amplifiedsimultaneously. Amplification can add sequencing adaptors to the nucleicacid.

In some embodiments, amplification can be performed on the substrate,for example, with bridge amplification. cDNAs can be homopolymer tailedin order to generate a compatible end for bridge amplification usingoligo(dT) probes on the substrate. In bridge amplification, the primerthat is complementary to the 3′ end of the template nucleic acid can bethe first primer of each pair that is covalently attached to the solidparticle. When a sample containing the template nucleic acid iscontacted with the particle and a single thermal cycle is performed, thetemplate molecule can be annealed to the first primer and the firstprimer is elongated in the forward direction by addition of nucleotidesto form a duplex molecule consisting of the template molecule and anewly formed DNA strand that is complementary to the template. In theheating step of the next cycle, the duplex molecule can be denatured,releasing the template molecule from the particle and leaving thecomplementary DNA strand attached to the particle through the firstprimer. In the annealing stage of the annealing and elongation step thatfollows, the complementary strand can hybridize to the second primer,which is complementary to a segment of the complementary strand at alocation removed from the first primer. This hybridization can cause thecomplementary strand to form a bridge between the first and secondprimers secured to the first primer by a covalent bond and to the secondprimer by hybridization. In the elongation stage, the second primer canbe elongated in the reverse direction by the addition of nucleotides inthe same reaction mixture, thereby converting the bridge to adouble-stranded bridge. The next cycle then begins, and thedouble-stranded bridge can be denatured to yield two single-strandednucleic acid molecules, each having one end attached to the particlesurface via the first and second primers, respectively, with the otherend of each unattached. In the annealing and elongation step of thissecond cycle, each strand can hybridize to a further complementaryprimer, previously unused, on the same particle, to form newsingle-strand bridges. The two previously unused primers that are nowhybridized elongate to convert the two new bridges to double-strandbridges.

The amplification reactions can comprise amplifying at least 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 100% of theplurality of nucleic acids.

Amplification of the labeled nucleic acids can comprise PCR-basedmethods or non-PCR based methods. Amplification of the labeled nucleicacids can comprise exponential amplification of the labeled nucleicacids. Amplification of the labeled nucleic acids can comprise linearamplification of the labeled nucleic acids. Amplification can beperformed by polymerase chain reaction (PCR). PCR can refer to areaction for the in vitro amplification of specific DNA sequences by thesimultaneous primer extension of complementary strands of DNA. PCR canencompass derivative forms of the reaction, including but not limitedto, RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplexedPCR, digital PCR, suppression PCR, semi-suppressive PCR and assemblyPCR.

In some embodiments, amplification of the labeled nucleic acidscomprises non-PCR based methods. Examples of non-PCR based methodsinclude, but are not limited to, multiple displacement amplification(MDA), transcription-mediated amplification (TMA), nucleic acidsequence-based amplification (NASBA), strand displacement amplification(SDA), real-time SDA, rolling circle amplification, or circle-to-circleamplification. Other non-PCR-based amplification methods includemultiple cycles of DNA-dependent RNA polymerase-driven RNA transcriptionamplification or RNA-directed DNA synthesis and transcription to amplifyDNA or RNA targets, a ligase chain reaction (LCR), a Qβ replicase (Qβ),use of palindromic probes, strand displacement amplification,oligonucleotide-driven amplification using a restriction endonuclease,an amplification method in which a primer is hybridized to a nucleicacid sequence and the resulting duplex is cleaved prior to the extensionreaction and amplification, strand displacement amplification using anucleic acid polymerase lacking 5′ exonuclease activity, rolling circleamplification, and/or ramification extension amplification (RAM).

In some embodiments, the methods disclosed herein further compriseconducting a nested polymerase chain reaction on the amplified amplicon(e.g., target). The amplicon can be double-stranded molecule. Thedouble-stranded molecule can comprise a double-stranded RNA molecule, adouble-stranded DNA molecule, or a RNA molecule hybridized to a DNAmolecule. One or both of the strands of the double-stranded molecule cancomprise a sample tag or molecular identifier label. Alternatively, theamplicon can be a single-stranded molecule. The single-stranded moleculecan comprise DNA, RNA, or a combination thereof. The nucleic acids ofthe present invention can comprise synthetic or altered nucleic acids.

In some embodiments, the method comprises repeatedly amplifying thelabeled nucleic acid to produce multiple amplicons. The methodsdisclosed herein can comprise conducting at least about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amplificationreactions. Alternatively, the method comprises conducting at least about25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100amplification reactions.

Amplification can further comprise adding one or more control nucleicacids to one or more samples comprising a plurality of nucleic acids.Amplification can further comprise adding one or more control nucleicacids to a plurality of nucleic acids. The control nucleic acids cancomprise a control label.

Amplification can comprise use of one or more non-natural nucleotides.Non-natural nucleotides can comprise photolabile and/or triggerablenucleotides. Examples of non-natural nucleotides include, but are notlimited to, peptide nucleic acid (PNA), morpholine and locked nucleicacid (LNA), as well as glycol nucleic acid (GNA) and threose nucleicacid (TNA). Non-natural nucleotides can be added to one or more cyclesof an amplification reaction. The addition of the non-naturalnucleotides can be used to identify products as specific cycles or timepoints in the amplification reaction.

Conducting the one or more amplification reactions can comprise the useof one or more primers. The one or more primers can comprise one or moreoligonucleotides. The one or more oligonucleotides can comprise at leastabout 7-9 nucleotides. The one or more oligonucleotides can compriseless than 12-15 nucleotides. The one or more primers can anneal to atleast a portion of the plurality of labeled nucleic acids. The one ormore primers can anneal to the 3′ end and/or 5′ end of the plurality oflabeled nucleic acids. The one or more primers can anneal to an internalregion of the plurality of labeled nucleic acids. The internal regioncan be at least about 50, 100, 150, 200, 220, 230, 240, 250, 260, 270,280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410,420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550,560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900 or 1000nucleotides from the 3′ ends the plurality of labeled nucleic acids. Theone or more primers can comprise a fixed panel of primers. The one ormore primers can comprise at least one or more custom primers. The oneor more primers can comprise at least one or more control primers. Theone or more primers can comprise at least one or more housekeeping geneprimers. The one or more primers can comprise a universal primer. Theuniversal primer can anneal to a universal primer binding site. The oneor more custom primers can anneal to the first sample tag, the secondsample tag, the molecular identifier label, the nucleic acid or aproduct thereof. The one or more primers can comprise a universal primerand a custom primer. The custom primer can be designed to amplify one ormore target nucleic acids. The target nucleic acids can comprise asubset of the total nucleic acids in one or more samples. In someembodiments, the primers are the probes attached to the array of thedisclosure.

In some embodiments, barcoding (e.g., stochastically barcoding) theplurality of targets in the sample further comprises generating anindexed library of the barcoded targets (e.g., stochastically barcodedtargets) or barcoded fragments of the targets. The barcode sequences ofdifferent barcodes (e.g., the molecular labels of different stochasticbarcodes) can be different from one another. Generating an indexedlibrary of the barcoded targets includes generating a plurality ofindexed polynucleotides from the plurality of targets in the sample. Forexample, for an indexed library of the barcoded targets comprising afirst indexed target and a second indexed target, the label region ofthe first indexed polynucleotide can differ from the label region of thesecond indexed polynucleotide by, by about, by at least, or by at most,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or a number or a rangebetween any two of these values, nucleotides. In some embodiments,generating an indexed library of the barcoded targets includescontacting a plurality of targets, for example mRNA molecules, with aplurality of oligonucleotides including a poly(T) region and a labelregion; and conducting a first strand synthesis using a reversetranscriptase to produce single-strand labeled cDNA molecules eachcomprising a cDNA region and a label region, wherein the plurality oftargets includes at least two mRNA molecules of different sequences andthe plurality of oligonucleotides includes at least two oligonucleotidesof different sequences. Generating an indexed library of the barcodedtargets can further comprise amplifying the single-strand labeled cDNAmolecules to produce double-strand labeled cDNA molecules; andconducting nested PCR on the double-strand labeled cDNA molecules toproduce labeled amplicons. In some embodiments, the method can includegenerating an adaptor-labeled amplicon.

Barcoding (e.g., stochastic barcoding) can include using nucleic acidbarcodes or tags to label individual nucleic acid (e.g., DNA or RNA)molecules. In some embodiments, it involves adding DNA barcodes or tagsto cDNA molecules as they are generated from mRNA. Nested PCR can beperformed to minimize PCR amplification bias. Adaptors can be added forsequencing using, for example, next generation sequencing (NGS). Thesequencing results can be used to determine cell labels, molecularlabels, and sequences of nucleotide fragments of the one or more copiesof the targets, for example at block 232 of FIG. 2.

FIG. 3 is a schematic illustration showing a non-limiting exemplaryprocess of generating an indexed library of the barcoded targets (e.g.,stochastically barcoded targets), such as barcoded mRNAs or fragmentsthereof. As shown in step 1, the reverse transcription process canencode each mRNA molecule with a unique molecular label, a cell label,and a universal PCR site. In particular, RNA molecules 302 can bereverse transcribed to produce labeled cDNA molecules 304, including acDNA region 306, by hybridization (e.g., stochastic hybridization) of aset of barcodes (e.g., stochastic barcodes) 310 to the poly(A) tailregion 308 of the RNA molecules 302. Each of the barcodes 310 cancomprise a target-binding region, for example a poly(dT) region 312, alabel region 314 (e.g., a barcode sequence or a molecule), and auniversal PCR region 316.

In some embodiments, the cell label can include 3 to 20 nucleotides. Insome embodiments, the molecular label can include 3 to 20 nucleotides.In some embodiments, each of the plurality of stochastic barcodesfurther comprises one or more of a universal label and a cell label,wherein universal labels are the same for the plurality of stochasticbarcodes on the solid support and cell labels are the same for theplurality of stochastic barcodes on the solid support. In someembodiments, the universal label can include 3 to 20 nucleotides. Insome embodiments, the cell label comprises 3 to 20 nucleotides.

In some embodiments, the label region 314 can include a barcode sequenceor a molecular label 318 and a cell label 320. In some embodiments, thelabel region 314 can include one or more of a universal label, adimension label, and a cell label. The barcode sequence or molecularlabel 318 can be, can be about, can be at least, or can be at most, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or anumber or a range between any of these values, of nucleotides in length.The cell label 320 can be, can be about, can be at least, or can be atmost, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, or a number or a range between any of these values, of nucleotidesin length. The universal label can be, can be about, can be at least, orcan be at most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70,80, 90, 100, or a number or a range between any of these values, ofnucleotides in length. Universal labels can be the same for theplurality of stochastic barcodes on the solid support and cell labelsare the same for the plurality of stochastic barcodes on the solidsupport. The dimension label can be, can be about, can be at least, orcan be at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70,80, 90, 100, or a number or a range between any of these values, ofnucleotides in length.

In some embodiments, the label region 314 can comprise, comprise about,comprise at least, or comprise at most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800,900, 1000, or a number or a range between any of these values, differentlabels, such as a barcode sequence or a molecular label 318 and a celllabel 320. Each label can be, can be about, can be at least, or can beat most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, or a number or a range between any of these values, of nucleotidesin length. A set of barcodes or stochastic barcodes 310 can contain,contain about, contain at least, or can be at most, 10, 20, 40, 50, 70,80, 90, 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³,10¹⁴, 10¹⁵, 10²⁰, or a number or a range between any of these values,barcodes or stochastic barcodes 310. And the set of barcodes orstochastic barcodes 310 can, for example, each contain a unique labelregion 314. The labeled cDNA molecules 304 can be purified to removeexcess barcodes or stochastic barcodes 310. Purification can compriseAmpure bead purification.

As shown in step 2, products from the reverse transcription process instep 1 can be pooled into 1 tube and PCR amplified with a 1^(st) PCRprimer pool and a 1^(st) universal PCR primer. Pooling is possiblebecause of the unique label region 314. In particular, the labeled cDNAmolecules 304 can be amplified to produce nested PCR labeled amplicons322. Amplification can comprise multiplex PCR amplification.Amplification can comprise a multiplex PCR amplification with 96multiplex primers in a single reaction volume. In some embodiments,multiplex PCR amplification can utilize, utilize about, utilize atleast, or utilize at most, 10, 20, 40, 50, 70, 80, 90, 10², 10³, 10⁴,10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹³, 10¹⁴, 10¹⁵, 10²⁰, or a number or arange between any of these values, multiplex primers in a singlereaction volume. Amplification can comprise using a 1^(st) PCR primerpool 324 comprising custom primers 326A-C targeting specific genes and auniversal primer 328. The custom primers 326 can hybridize to a regionwithin the cDNA portion 306′ of the labeled cDNA molecule 304. Theuniversal primer 328 can hybridize to the universal PCR region 316 ofthe labeled cDNA molecule 304.

As shown in step 3 of FIG. 3, products from PCR amplification in step 2can be amplified with a nested PCR primers pool and a 2^(nd) universalPCR primer. Nested PCR can minimize PCR amplification bias. Inparticular, the nested PCR labeled amplicons 322 can be furtheramplified by nested PCR. The nested PCR can comprise multiplex PCR withnested PCR primers pool 330 of nested PCR primers 332 a-c and a 2^(nd)universal PCR primer 328′ in a single reaction volume. The nested PCRprimer pool 328 can contain, contain about, contain at least, or containat most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or a number or arange between any of these values, different nested PCR primers 330. Thenested PCR primers 332 can contain an adaptor 334 and hybridize to aregion within the cDNA portion 306″ of the labeled amplicon 322. Theuniversal primer 328′ can contain an adaptor 336 and hybridize to theuniversal PCR region 316 of the labeled amplicon 322. Thus, step 3produces adaptor-labeled amplicon 338. In some embodiments, nested PCRprimers 332 and the 2^(nd) universal PCR primer 328′ may not contain theadaptors 334 and 336. The adaptors 334 and 336 can instead be ligated tothe products of nested PCR to produce adaptor-labeled amplicon 338.

As shown in step 4, PCR products from step 3 can be PCR amplified forsequencing using library amplification primers. In particular, theadaptors 334 and 336 can be used to conduct one or more additionalassays on the adaptor-labeled amplicon 338. The adaptors 334 and 336 canbe hybridized to primers 340 and 342. The one or more primers 340 and342 can be PCR amplification primers. The one or more primers 340 and342 can be sequencing primers. The one or more adaptors 334 and 336 canbe used for further amplification of the adaptor-labeled amplicons 338.The one or more adaptors 334 and 336 can be used for sequencing theadaptor-labeled amplicon 338. The primer 342 can contain a plate index344 so that amplicons generated using the same set of barcodes orstochastic barcodes 310 can be sequenced in one sequencing reactionusing next generation sequencing (NGS).

Compositions Comprising Cellular Component Binding Reagents Associatedwith Oligonucleotides

Some embodiments disclosed herein provide a plurality of compositionseach comprising a cellular component binding reagent (such as a proteinbinding reagent) that is conjugated with an oligonucleotide, wherein theoligonucleotide comprises a unique identifier for the cellular componentbinding reagent that it is conjugated with. Cellular component bindingreagents (such as barcoded antibodies) and their uses (such as sampleindexing of cells) have been described in U.S. Patent ApplicationPublication Nos. US2018/0088112 and US2018/0346970; the content of eachof these is incorporated herein by reference in its entirety.

In some embodiments, the cellular component binding reagent is capableof specifically binding to a cellular component target. For example, abinding target of the cellular component binding reagent can be, orcomprise, a carbohydrate, a lipid, a protein, an extracellular protein,a cell-surface protein, a cell marker, a B-cell receptor, a T-cellreceptor, a major histocompatibility complex, a tumor antigen, areceptor, an integrin, an intracellular protein, or any combinationthereof. In some embodiments, the cellular component binding reagent(e.g., a protein binding reagent) is capable of specifically binding toan antigen target or a protein target. In some embodiments, each of theoligonucleotides can comprise a barcode, such as a stochastic barcode. Abarcode can comprise a barcode sequence (e.g., a molecular label), acell label, a sample label, or any combination thereof. In someembodiments, each of the oligonucleotides can comprise a linker. In someembodiments, each of the oligonucleotides can comprise a binding sitefor an oligonucleotide probe, such as a poly(A) tail. For example, thepoly(A) tail can be, e.g., unanchored to a solid support or anchored toa solid support. The poly(A) tail can be from about 10 to 50 nucleotidesin length. In some embodiments, the poly(A) tail can be 18 nucleotidesin length. The oligonucleotides can comprise deoxyribonucleotides,ribonucleotides, or both.

The unique identifiers can be, for example, a nucleotide sequence havingany suitable length, for example, from about 4 nucleotides to about 200nucleotides. In some embodiments, the unique identifier is a nucleotidesequence of 25 nucleotides to about 45 nucleotides in length. In someembodiments, the unique identifier can have a length that is, is about,is less than, is greater than, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 70, 80, 90, 100, 200 nucleotides, or a range that isbetween any two of the above values.

In some embodiments, the unique identifiers are selected from a diverseset of unique identifiers. The diverse set of unique identifiers cancomprise, or comprise about, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200,300, 400, 500, 600, 700, 800, 900, 1000, 2000, 5000, or a number or arange between any two of these values, different unique identifiers. Thediverse set of unique identifiers can comprise at least, or comprise atmost, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700,800, 900, 1000, 2000, or 5000, different unique identifiers. In someembodiments, the set of unique identifiers is designed to have minimalsequence homology to the DNA or RNA sequences of the sample to beanalyzed. In some embodiments, the sequences of the set of uniqueidentifiers are different from each other, or the complement thereof,by, or by about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nucleotides, or a numberor a range between any two of these values. In some embodiments, thesequences of the set of unique identifiers are different from eachother, or the complement thereof, by at least, or by at most, 1, 2, 3,4, 5, 6, 7, 8, 9, or 10 nucleotides. In some embodiments, the sequencesof the set of unique identifiers are different from each other, or thecomplement thereof, by at least 3%, at least 5%, at least 8%, at least10%, at least 15%, at least 20%, or more.

In some embodiments, the unique identifiers can comprise a binding sitefor a primer, such as universal primer. In some embodiments, the uniqueidentifiers can comprise at least two binding sites for a primer, suchas a universal primer. In some embodiments, the unique identifiers cancomprise at least three binding sites for a primer, such as a universalprimer. The primers can be used for amplification of the uniqueidentifiers, for example, by PCR amplification. In some embodiments, theprimers can be used for nested PCR reactions.

Any suitable cellular component binding reagents are contemplated inthis disclosure, such as protein binding reagents, antibodies orfragments thereof, aptamers, small molecules, ligands, peptides,oligonucleotides, etc., or any combination thereof. In some embodiments,the cellular component binding reagents can be polyclonal antibodies,monoclonal antibodies, recombinant antibodies, single chain antibody(sc-Ab), or fragments thereof, such as Fab, Fv, etc. In someembodiments, the plurality of cellular component binding reagents cancomprise, or comprise about, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200,300, 400, 500, 600, 700, 800, 900, 1000, 2000, 5000, or a number or arange between any two of these values, different cellular componentreagents. In some embodiments, the plurality of cellular componentbinding reagents can comprise at least, or comprise at most, 20, 30, 40,50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000,2000, or 5000, different cellular component reagents.

The oligonucleotide can be conjugated with the cellular componentbinding reagent through various mechanism. In some embodiments, theoligonucleotide can be conjugated with the cellular component bindingreagent covalently. In some embodiment, the oligonucleotide can beconjugated with the cellular component binding reagent non-covalently.In some embodiments, the oligonucleotide is conjugated with the cellularcomponent binding reagent through a linker. The linker can be, forexample, cleavable or detachable from the cellular component bindingreagent and/or the oligonucleotide. In some embodiments, the linker cancomprise a chemical group that reversibly attaches the oligonucleotideto the cellular component binding reagents. The chemical group can beconjugated to the linker, for example, through an amine group. In someembodiments, the linker can comprise a chemical group that forms astable bond with another chemical group conjugated to the cellularcomponent binding reagent. For example, the chemical group can be a UVphotocleavable group, a disulfide bond, a streptavidin, a biotin, anamine, etc. In some embodiments, the chemical group can be conjugated tothe cellular component binding reagent through a primary amine on anamino acid, such as lysine, or the N-terminus. Commercially availableconjugation kits, such as the Protein-Oligo Conjugation Kit (Solulink,Inc., San Diego, Calif.), the Thunder-Link® oligo conjugation system(Innova Biosciences, Cambridge, United Kingdom), etc., can be used toconjugate the oligonucleotide to the cellular component binding reagent.

The oligonucleotide can be conjugated to any suitable site of thecellular component binding reagent (e.g., a protein binding reagent), aslong as it does not interfere with the specific binding between thecellular component binding reagent and its cellular component target. Insome embodiments, the cellular component binding reagent is a protein,such as an antibody. In some embodiments, the cellular component bindingreagent is not an antibody. In some embodiments, the oligonucleotide canbe conjugated to the antibody anywhere other than the antigen-bindingsite, for example, the Fc region, the C_(H)1 domain, the C_(H)2 domain,the C_(H)3 domain, the C_(L) domain, etc. Methods of conjugatingoligonucleotides to cellular component binding reagents (e.g.,antibodies) have been previously disclosed, for example, in U.S. Pat.No. 6,531,283, the content of which is hereby expressly incorporated byreference in its entirety. Stoichiometry of oligonucleotide to cellularcomponent binding reagent can be varied. To increase the sensitivity ofdetecting the cellular component binding reagent specificoligonucleotide in sequencing, it may be advantageous to increase theratio of oligonucleotide to cellular component binding reagent duringconjugation. In some embodiments, each cellular component bindingreagent can be conjugated with a single oligonucleotide molecule. Insome embodiments, each cellular component binding reagent can beconjugated with more than one oligonucleotide molecule, for example, atleast, or at most, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 1000, or anumber or a range between any two of these values, oligonucleotidemolecules wherein each of the oligonucleotide molecule comprises thesame, or different, unique identifiers. In some embodiments, eachcellular component binding reagent can be conjugated with more than oneoligonucleotide molecule, for example, at least, or at most, 2, 3, 4, 5,10, 20, 30, 40, 50, 100, 1000, oligonucleotide molecules, wherein eachof the oligonucleotide molecule comprises the same, or different, uniqueidentifiers.

In some embodiments, the plurality of cellular component bindingreagents are capable of specifically binding to a plurality of cellularcomponent targets in a sample, such as a single cell, a plurality ofcells, a tissue sample, a tumor sample, a blood sample, or the like. Insome embodiments, the plurality of cellular component targets comprisesa cell-surface protein, a cell marker, a B-cell receptor, a T-cellreceptor, an antibody, a major histocompatibility complex, a tumorantigen, a receptor, or any combination thereof. In some embodiments,the plurality of cellular component targets can comprise intracellularcellular components. In some embodiments, the plurality of cellularcomponent targets can comprise intracellular cellular components. Insome embodiments, the plurality of cellular components can be, or beabout, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95%, 98%, 99%, or a number or a range between any two ofthese values, of all the cellular components (e.g., proteins) in a cellor an organism. In some embodiments, the plurality of cellularcomponents can be at least, or be at most, 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%,of all the cellular components (e.g., proteins) in a cell or anorganism. In some embodiments, the plurality of cellular componenttargets can comprise, or comprise about, 2, 3, 4, 5, 10, 20, 30, 40, 50,100, 1000, 10000, or a number or a range between any two of thesevalues, different cellular component targets. In some embodiments, theplurality of cellular component targets can comprise at least, orcomprise at most, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 1000, 10000,different cellular component targets.

FIG. 4 shows a schematic illustration of an exemplary cellular componentbinding reagent (e.g., an antibody) that is associated (e.g.,conjugated) with an oligonucleotide comprising a unique identifiersequence for the antibody. An oligonucleotide-conjugated with a cellularcomponent binding reagent, an oligonucleotide for conjugation with acellular component binding reagent, or an oligonucleotide previouslyconjugated with a cellular component binding reagent can be referred toherein as an antibody oligonucleotide (abbreviated as a binding reagentoligonucleotide). An oligonucleotide-conjugated with an antibody, anoligonucleotide for conjugation with an antibody, or an oligonucleotidepreviously conjugated with an antibody can be referred to herein as anantibody oligonucleotide (abbreviated as an “AbOligo” or “AbO”). Theoligonucleotide can also comprise additional components, including butnot limited to, one or more linker, one or more unique identifier forthe antibody, optionally one or more barcode sequences (e.g., molecularlabels), and a poly(A) tail. In some embodiments, the oligonucleotidecan comprise, from 5′ to 3′, a linker, a unique identifier, a barcodesequence (e.g., a molecular label), and a poly(A) tail. An antibodyoligonucleotide can be an mRNA mimic.

FIG. 5 shows a schematic illustration of an exemplary cellular componentbinding reagent (e.g., an antibody) that is associated (e.g.,conjugated) with an oligonucleotide comprising a unique identifiersequence for the antibody. The cellular component binding reagent can becapable of specifically binding to at least one cellular componenttarget, such as an antigen target or a protein target. A binding reagentoligonucleotide (e.g., a sample indexing oligonucleotide, or an antibodyoligonucleotide) can comprise a sequence (e.g., a sample indexingsequence) for performing the methods of the disclosure. For example, asample indexing oligonucleotide can comprise a sample indexing sequencefor identifying sample origin of one or more cells of a sample. Indexingsequences (e.g., sample indexing sequences) of at least two compositionscomprising two cellular component binding reagents (e.g., sampleindexing compositions) of the plurality of compositions comprisingcellular component binding reagents can comprise different sequences. Insome embodiments, the binding reagent oligonucleotide is not homologousto genomic sequences of a species. The binding reagent oligonucleotidecan be configured to be (or can be) detachable or non-detachable fromthe cellular component binding reagent.

The oligonucleotide conjugated to a cellular component binding reagentcan, for example, comprise a barcode sequence (e.g., a molecular labelsequence), a poly(A) tail, or a combination thereof. An oligonucleotideconjugated to a cellular component binding reagent can be an mRNA mimic.In some embodiments, the sample indexing oligonucleotide comprises asequence complementary to a capture sequence of at least one barcode ofthe plurality of barcodes. A target binding region of the barcode cancomprise the capture sequence. The target binding region can, forexample, comprise a poly(dT) region. In some embodiments, the sequenceof the sample indexing oligonucleotide complementary to the capturesequence of the barcode can comprise a poly(A) tail. The sample indexingoligonucleotide can comprise a molecular label.

In some embodiments, the binding reagent oligonucleotide (e.g., thesample oligonucleotide) comprises a nucleotide sequence of, or anucleotide sequence of about, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120,128, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250,260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390,400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530,540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670,680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810,820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950,960, 970, 980, 990, 1000, or a number or a range between any two ofthese values, nucleotides in length. In some embodiments, the bindingreagent oligonucleotide comprises a nucleotide sequence of at least, orof at most, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25,30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 128, 130, 140, 150,160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290,300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430,440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570,580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710,720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850,860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or1000, nucleotides in length.

In some embodiments, the cellular component binding reagent comprises anantibody, a tetramer, an aptamer, a protein scaffold, or a combinationthereof. The binding reagent oligonucleotide can be conjugated to thecellular component binding reagent, for example, through a linker. Thebinding reagent oligonucleotide can comprise the linker. The linker cancomprise a chemical group. The chemical group can be reversibly, orirreversibly, attached to the molecule of the cellular component bindingreagent. The chemical group can be selected from the group consisting ofa UV photocleavable group, a disulfide bond, a streptavidin, a biotin,an amine, and any combination thereof.

In some embodiments, the cellular component binding reagent can bind toADAM10, CD156c, ANO6, ATP1B2, ATP1B3, BSG, CD147, CD109, CD230, CD29,CD298, ATP1B3, CD44, CD45, CD47, CD51, CD59, CD63, CD97, CD98, SLC3A2,CLDND1, HLA-ABC, ICAM1, ITFG3, MPZL1, NA K ATPase alpha1, ATP1A1, NPTN,PMCA ATPase, ATP2B1, SLC1A5, SLC29A1, SLC2A1, SLC44A2, or anycombination thereof.

In some embodiments, the protein target is, or comprises, anextracellular protein, an intracellular protein, or any combinationthereof. In some embodiments, the antigen or protein target is, orcomprises, a cell-surface protein, a cell marker, a B-cell receptor, aT-cell receptor, a major histocompatibility complex, a tumor antigen, areceptor, an integrin, or any combination thereof. The antigen orprotein target can be, or comprise, a lipid, a carbohydrate, or anycombination thereof. The protein target can be selected from a groupcomprising a number of protein targets. The number of antigen target orprotein targets can be, or be about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, or a numberor a range between any two of these values. The number of proteintargets can be at least, or be at most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800,900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000.

The cellular component binding reagent (e.g., a protein binding reagent)can be associated with two or more binding reagent oligonucleotide(e.g., sample indexing oligonucleotides) with an identical sequence. Thecellular component binding reagent can be associated with two or morebinding reagent oligonucleotides with different sequences. The number ofbinding reagent oligonucleotides associated with the cellular componentbinding reagent can be different in different implementations. In someembodiments, the number of binding reagent oligonucleotides, whetherhaving an identical sequence, or different sequences, can be, or beabout, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or a number or arange between any two of these values. In some embodiments, the numberof binding reagent oligonucleotides can be at least, or be at most, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200,300, 400, 500, 600, 700, 800, 900, or 1000.

The plurality of compositions comprising cellular component bindingreagents (e.g., the plurality of sample indexing compositions) cancomprise one or more additional cellular component binding reagents notconjugated with the binding reagent oligonucleotide (such as sampleindexing oligonucleotide), which is also referred to herein as thebinding reagent oligonucleotide-free cellular component binding reagent(such as sample indexing oligonucleotide-free cellular component bindingreagent). The number of additional cellular component binding reagentsin the plurality of compositions can be different in differentimplementations. In some embodiments, the number of additional cellularcomponent binding reagents can be, or be about, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or a number or a rangebetween any two of these values. In some embodiments, the number ofadditional cellular component binding reagents can be at least, or be atmost, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or100. The cellular component binding reagent and any of the additionalcellular component binding reagents can be identical, in someembodiments.

In some embodiments, a mixture comprising cellular component bindingreagent(s) that is conjugated with one or more binding reagentoligonucleotides (e.g., sample indexing oligonucleotides) and cellularcomponent binding reagent(s) that is not conjugated with binding reagentoligonucleotides is provided. The mixture can be used in someembodiments of the methods disclosed herein, for example, to contact thesample(s) and/or cell(s). The ratio of (1) the number of a cellularcomponent binding reagent conjugated with a binding reagentoligonucleotide and (2) the number of another cellular component bindingreagent (e.g., the same cellular component binding reagent) notconjugated with the binding reagent oligonucleotide (e.g., sampleindexing oligonucleotide) or other binding reagent oligonucleotide(s) inthe mixture can be different in different implementations. In someembodiments, the ratio can be, or be about, 1:1, 1:1.1, 1:1.2, 1:1.3,1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.5, 1:3, 1:4, 1:5,1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17,1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29,1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, 1:41,1:42, 1:43, 1:44, 1:45, 1:46, 1:47, 1:48, 1:49, 1:50, 1:51, 1:52, 1:53,1:54, 1:55, 1:56, 1:57, 1:58, 1:59, 1:60, 1:61, 1:62, 1:63, 1:64, 1:65,1:66, 1:67, 1:68, 1:69, 1:70, 1:71, 1:72, 1:73, 1:74, 1:75, 1:76, 1:77,1:78, 1:79, 1:80, 1:81, 1:82, 1:83, 1:84, 1:85, 1:86, 1:87, 1:88, 1:89,1:90, 1:91, 1:92, 1:93, 1:94, 1:95, 1:96, 1:97, 1:98, 1:99, 1:100,1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, 1:2000,1:3000, 1:4000, 1:5000, 1:6000, 1:7000, 1:8000, 1:9000, 1:10000, or anumber or a range between any two of the values. In some embodiments,the ratio can be at least, or be at most, 1:1, 1:1.1, 1:1.2, 1:1.3,1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.5, 1:3, 1:4, 1:5,1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17,1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29,1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, 1:41,1:42, 1:43, 1:44, 1:45, 1:46, 1:47, 1:48, 1:49, 1:50, 1:51, 1:52, 1:53,1:54, 1:55, 1:56, 1:57, 1:58, 1:59, 1:60, 1:61, 1:62, 1:63, 1:64, 1:65,1:66, 1:67, 1:68, 1:69, 1:70, 1:71, 1:72, 1:73, 1:74, 1:75, 1:76, 1:77,1:78, 1:79, 1:80, 1:81, 1:82, 1:83, 1:84, 1:85, 1:86, 1:87, 1:88, 1:89,1:90, 1:91, 1:92, 1:93, 1:94, 1:95, 1:96, 1:97, 1:98, 1:99, 1:100,1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, 1:2000,1:3000, 1:4000, 1:5000, 1:6000, 1:7000, 1:8000, 1:9000, or 1:10000.

In some embodiments, the ratio can be, or be about, 1:1, 1.1:1, 1.2:1,1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.5:1, 3:1, 4:1,5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1,18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1,30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1,42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1, 53:1,54:1, 55:1, 56:1, 57:1, 58:1, 59:1, 60:1, 61:1, 62:1, 63:1, 64:1, 65:1,66:1, 67:1, 68:1, 69:1, 70:1, 71:1, 72:1, 73:1, 74:1, 75:1, 76:1, 77:1,78:1, 79:1, 80:1, 81:1, 82:1, 83:1, 84:1, 85:1, 86:1, 87:1, 88:1, 89:1,90:1, 91:1, 92:1, 93:1, 94:1, 95:1, 96:1, 97:1, 98:1, 99:1, 100:1,200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, 1000:1, 2000:1,3000:1, 4000:1, 5000:1, 6000:1, 7000:1, 8000:1, 9000:1, 10000:1, or anumber or a range between any two of the values. In some embodiments,the ratio can be at least, or be at most, 1:1, 1.1:1, 1.2:1, 1.3:1,1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.5:1, 3:1, 4:1, 5:1,6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1,18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1,30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1,42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1, 53:1,54:1, 55:1, 56:1, 57:1, 58:1, 59:1, 60:1, 61:1, 62:1, 63:1, 64:1, 65:1,66:1, 67:1, 68:1, 69:1, 70:1, 71:1, 72:1, 73:1, 74:1, 75:1, 76:1, 77:1,78:1, 79:1, 80:1, 81:1, 82:1, 83:1, 84:1, 85:1, 86:1, 87:1, 88:1, 89:1,90:1, 91:1, 92:1, 93:1, 94:1, 95:1, 96:1, 97:1, 98:1, 99:1, 100:1,200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, 1000:1, 2000:1,3000:1, 4000:1, 5000:1, 6000:1, 7000:1, 8000:1, 9000:1, or 10000:1.

A cellular component binding reagent can be conjugated with a bindingreagent oligonucleotide (e.g., a sample indexing oligonucleotide), ornot. In some embodiments, the percentage of the cellular componentbinding reagent conjugated with a binding reagent oligonucleotide (e.g.,a sample indexing oligonucleotide) in a mixture comprising the cellularcomponent binding reagent that is conjugated with the binding reagentoligonucleotide and the cellular component binding reagent(s) that isnot conjugated with the binding reagent oligonucleotide can be, or beabout, 0.000000001%, 0.00000001%, 0.0000001%, 0.000001%, 0.00001%,0.0001%, 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%,25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%,39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two ofthese values. In some embodiments, the percentage of the cellularcomponent binding reagent conjugated with a sample indexingoligonucleotide in a mixture can be at least, or be at most,0.000000001%, 0.00000001%, 0.0000001%, 0.000001%, 0.00001%, 0.0001%,0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%,13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%,27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%,41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%.

In some embodiments, the percentage of the cellular component bindingreagent not conjugated with a binding reagent oligonucleotide (e.g., asample indexing oligonucleotide) in a mixture comprising a cellularcomponent binding reagent conjugated with a binding reagentoligonucleotide (e.g., a sample indexing oligonucleotide) and thecellular component binding reagent that is not conjugated with thesample indexing oligonucleotide can be, or be about, 0.000000001%,0.00000001%, 0.0000001%, 0.000001%, 0.00001%, 0.0001%, 0.001%, 0.01%,0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%,30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%,44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,100%, or a number or a range between any two of these values. In someembodiments, the percentage of the cellular component binding reagentnot conjugated with a binding reagent oligonucleotide in a mixture canbe at least, or be at most, 0.000000001%, 0.00000001%, 0.0000001%,0.000001%, 0.00001%, 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%,21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%,35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%,49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

Cellular Component Cocktails

In some embodiments, a cocktail of cellular component binding reagents(e.g., an antibody cocktail) can be used to increase labelingsensitivity in the methods disclosed herein. Without being bound by anyparticular theory, it is believed that this may be because cellularcomponent expression or protein expression can vary between cell typesand cell states, making finding a universal cellular component bindingreagent or antibody that labels all cell types challenging. For example,cocktail of cellular component binding reagents can be used to allow formore sensitive and efficient labeling of more sample types. The cocktailof cellular component binding reagents can include two or more differenttypes of cellular component binding reagents, for example a wider rangeof cellular component binding reagents or antibodies. Cellular componentbinding reagents that label different cellular component targets can bepooled together to create a cocktail that sufficiently labels all celltypes, or one or more cell types of interest.

In some embodiments, each of the plurality of compositions (e.g., sampleindexing compositions) comprises a cellular component binding reagent.In some embodiments, a composition of the plurality of compositionscomprises two or more cellular component binding reagents, wherein eachof the two or more cellular component binding reagents is associatedwith a binding reagent oligonucleotide (e.g., a sample indexingoligonucleotide), wherein at least one of the two or more cellularcomponent binding reagents is capable of specifically binding to atleast one of the one or more cellular component targets. The sequencesof the binding reagent oligonucleotides associated with the two or morecellular component binding reagents can be identical. The sequences ofthe binding reagent oligonucleotides associated with the two or morecellular component binding reagents can comprise different sequences.Each of the plurality of compositions can comprise the two or morecellular component binding reagents.

The number of different types of cellular component binding reagents(e.g., a CD147 antibody and a CD47 antibody) in a composition can bedifferent in different implementations. A composition with two or moredifferent types of cellular component binding reagents can be referredto herein as a cellular component binding reagent cocktail (e.g., asample indexing composition cocktail). The number of different types ofcellular component binding reagents in a cocktail can vary. In someembodiments, the number of different types of cellular component bindingreagents in cocktail can be, or be about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 10000, 100000, or a number or a range between any two of thesevalues. In some embodiments, the number of different types of cellularcomponent binding reagents in cocktail can be at least, or be at most,2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200,300, 400, 500, 600, 700, 800, 900, 1000, 10000, or 100000. The differenttypes of cellular component binding reagents can be conjugated tobinding reagent oligonucleotides with the same or different sequences(e.g., sample indexing sequences).

Methods of Quantitative Analysis of Cellular Component Targets

In some embodiments, the methods disclosed herein can also be used forquantitative analysis of a plurality of cellular component targets (forexample, protein targets) in a sample using the compositions disclosedherein and oligonucleotide probes that can associate a barcode sequence(e.g., a molecular label sequence) to the oligonucleotides of thecellular component binding reagents (e.g., protein binding reagents).The oligonucleotides of the cellular component binding reagents can be,or comprise, an antibody oligonucleotide, a sample indexingoligonucleotide, a cell identification oligonucleotide, a controlparticle oligonucleotide, a control oligonucleotide, an interactiondetermination oligonucleotide, etc. In some embodiments, the sample canbe a single cell, a plurality of cells, a tissue sample, a tumor sample,a blood sample, or the like. In some embodiments, the sample cancomprise a mixture of cell types, such as normal cells, tumor cells,blood cells, B cells, T cells, maternal cells, fetal cells, etc., or amixture of cells from different subjects.

In some embodiments, the sample can comprise a plurality of single cellsseparated into individual compartments, such as microwells in amicrowell array.

In some embodiments, the binding target of the plurality of cellularcomponent target (i.e., the cellular component target) can be, orcomprise, a carbohydrate, a lipid, a protein, an extracellular protein,a cell-surface protein, a cell marker, a B-cell receptor, a T-cellreceptor, a major histocompatibility complex, a tumor antigen, areceptor, an integrin, an intracellular protein, or any combinationthereof. In some embodiments, the cellular component target is a proteintarget. In some embodiments, the plurality of cellular component targetscomprises a cell-surface protein, a cell marker, a B-cell receptor, aT-cell receptor, an antibody, a major histocompatibility complex, atumor antigen, a receptor, or any combination thereof. In someembodiments, the plurality of cellular component targets can compriseintracellular cellular components. In some embodiments, the plurality ofcellular components can be at least 1%, at least 2%, at least 3%, atleast 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least9%, at least 10%, at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 98%, at least 99%, or more, of all the encoded cellularcomponents in an organism. In some embodiments, the plurality ofcellular component targets can comprise at least 2, at least 3, at least4, at least 5, at least 10, at least 20, at least 30, at least 40, atleast 50, at least 100, at least 1000, at least 10000, or more differentcellular component targets.

In some embodiments, the plurality of cellular component bindingreagents is contacted with the sample for specific binding with theplurality of cellular component targets. Unbound cellular componentbinding reagents can be removed, for example, by washing. In embodimentswhere the sample comprises cells, any cellular component bindingreagents not specifically bound to the cells can be removed.

In some instances, cells from a population of cells can be separated(e.g., isolated) into wells of a substrate of the disclosure. Thepopulation of cells can be diluted prior to separating. The populationof cells can be diluted such that at least 1%, 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or100%, of wells of the substrate receive a single cell. The population ofcells can be diluted such that at most 1%, 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or100%, of wells of the substrate receive a single cell. The population ofcells can be diluted such that the number of cells in the dilutedpopulation is, or is at least, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, ofthe number of wells on the substrate. The population of cells can bediluted such that the number of cells in the diluted population is, oris at least, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, of the number of wellson the substrate. In some instances, the population of cells is dilutedsuch that the number of cells is about 10% of the number of wells in thesubstrate.

Distribution of single cells into wells of the substrate can follow aPoisson distribution. For example, there can be at least a 0.1%, 0.5%,1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, or more probability that awell of the substrate has more than one cell. There can be at least a0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, or moreprobability that a well of the substrate has more than one cell.Distribution of single cells into wells of the substrate can be random.Distribution of single cells into wells of the substrate can benon-random. The cells can be separated such that a well of the substratereceives only one cell.

In some embodiments, the cellular component binding reagents can beadditionally conjugated with fluorescent molecules to enable flowsorting of cells into individual compartments.

In some embodiments, the methods disclosed herein provide contacting aplurality of compositions with the sample for specific binding with theplurality of cellular component targets. It would be appreciated thatthe conditions used may allow specific binding of the cellular componentbinding reagents, e.g., antibodies, to the cellular component targets.Following the contacting step, unbound compositions can be removed. Forexample, in embodiments where the sample comprises cells, and thecompositions specifically bind to cellular component targets arecell-surface cellular components, such as cell-surface proteins, unboundcompositions can be removed by washing the cells with buffer such thatonly compositions that specifically bind to the cellular componenttargets remain with the cells.

In some embodiments, the methods disclosed herein can compriseassociating an oligonucleotide (e.g., a barcode, or a stochasticbarcode), including a barcode sequence (such as a molecular label), acell label, a sample label, etc., or any combination thereof, to theplurality of oligonucleotides associated with the cellular componentbinding reagents. For example, a plurality of oligonucleotide probescomprising a barcode can be used to hybridize to the plurality ofoligonucleotides of the compositions.

In some embodiments, the plurality of oligonucleotide probes can beimmobilized on solid supports. The solid supports can be free floating,e.g., beads in a solution. The solid supports can be embedded in asemi-solid or solid array. In some embodiments, the plurality ofoligonucleotide probes may not be immobilized on solid supports. Whenthe plurality of oligonucleotide probes are in close proximity to theplurality associated with oligonucleotides of the cellular componentbinding reagents, the plurality of oligonucleotides of the cellularcomponent binding reagents can hybridize to the oligonucleotide probes.The oligonucleotide probes can be contacted at a non-depletable ratiosuch that each distinct oligonucleotide of the cellular componentbinding reagents can associate with oligonucleotide probes havingdifferent barcode sequences (e.g., molecular labels) of the disclosure.

In some embodiments, the methods disclosed herein provide detaching theoligonucleotides from the cellular component binding reagents that arespecifically bound to the cellular component targets. Detachment can beperformed in a variety of ways to separate the chemical group from thecellular component binding reagent, such as UV photocleaving, chemicaltreatment (e.g., dithiothreitol treatment), heating, enzyme treatment,or any combination thereof. Detaching the oligonucleotide from thecellular component binding reagent can be performed either before,after, or during the step of hybridizing the plurality ofoligonucleotide probes to the plurality of oligonucleotides of thecompositions.

Methods of Simultaneous Quantitative Analysis of Cellular Component andNucleic Acid Targets

In some embodiments, the methods disclosed herein can also be used forsimultaneous quantitative analysis of a plurality of cellular componenttargets (e.g., protein targets) and a plurality of nucleic acid targetmolecules in a sample using the compositions disclosed herein andoligonucleotide probes that can associate a barcode sequence (e.g., amolecular label sequence) to both the oligonucleotides of the cellularcomponent binding reagents and nucleic acid target molecules. Othermethods of simultaneous quantitative analysis of a plurality of cellularcomponent targets and a plurality of nucleic acid target molecules aredescribed in U.S. Patent Application Publication No. US2018/0088112 andU.S. Patent Application Publication No. US2018/0346970; the content ofeach of these is incorporated herein by reference in its entirety. Insome embodiments, the sample can be a single cell, a plurality of cells,a tissue sample, a tumor sample, a blood sample, or the like. In someembodiments, the sample can comprise a mixture of cell types, such asnormal cells, tumor cells, blood cells, B cells, T cells, maternalcells, fetal cells, or a mixture of cells from different subjects.

In some embodiments, the sample can comprise a plurality of single cellsseparated into individual compartments, such as microwells in amicrowell array.

In some embodiments, the plurality of cellular component targetscomprises a cell-surface protein, a cell marker, a B-cell receptor, aT-cell receptor, an antibody, a major histocompatibility complex, atumor antigen, a receptor, or any combination thereof. In someembodiments, the plurality of cellular component targets can compriseintracellular cellular components. In some embodiments, the plurality ofcellular components can be, or be about, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or anumber or a range between any two of these values, of all the cellularcomponents, such as expressed proteins, in an organism, or one or morecells of the organism. In some embodiments, the plurality of cellularcomponents can be at least, or be at most, 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%,of all the cellular components, such as proteins could be expressed, inan organism, or one or more cells of the organism. In some embodiments,the plurality of cellular component targets can comprise, or compriseabout, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 1000, 10000, or a number ora range between any two of these values, different cellular componenttargets. In some embodiments, the plurality of cellular componenttargets can comprise at least, or comprise at most, 2, 3, 4, 5, 10, 20,30, 40, 50, 100, 1000, or 10000, different cellular component targets.

In some embodiments, the plurality of cellular component bindingreagents is contacted with the sample for specific binding with theplurality of cellular component targets. Unbound cellular componentbinding reagents can be removed, for example, by washing. In embodimentswhere the sample comprises cells, any cellular component bindingreagents not specifically bound to the cells can be removed.

In some instances, cells from a population of cells can be separated(e.g., isolated) into wells of a substrate of the disclosure. Thepopulation of cells can be diluted prior to separating. The populationof cells can be diluted such that at least 1%, 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or100% of wells of the substrate receive a single cell. The population ofcells can be diluted such that at most 1%, 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%of wells of the substrate receive a single cell. The population of cellscan be diluted such that the number of cells in the diluted populationis, or is at least, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the number ofwells on the substrate. The population of cells can be diluted such thatthe number of cells in the diluted population is, or is at least, 1%,5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, or 100% of the number of wells on thesubstrate. In some instances, the population of cells is diluted suchthat the number of cell is about 10% of the number of wells in thesubstrate.

Distribution of single cells into wells of the substrate can follow aPoisson distribution. For example, there can be at least a 0.1%, 0.5%,1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, or more probability that awell of the substrate has more than one cell. There can be at least a0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, or moreprobability that a well of the substrate has more than one cell.Distribution of single cells into wells of the substrate can be random.Distribution of single cells into wells of the substrate can benon-random. The cells can be separated such that a well of the substratereceives only one cell.

In some embodiments, the cellular component binding reagents can beadditionally conjugated with fluorescent molecules to enable flowsorting of cells into individual compartments.

In some embodiments, the methods disclosed herein provide contacting aplurality of compositions with the sample for specific binding with theplurality of cellular component targets. It would be appreciated thatthe conditions used may allow specific binding of the cellular componentbinding reagents, e.g., antibodies, to the cellular component targets.Following the contacting step, unbound compositions can be removed. Forexample, in embodiments where the sample comprises cells, and thecompositions specifically bind to cellular component targets are on thecell surface, such as cell-surface proteins, unbound compositions can beremoved by washing the cells with buffer such that only compositionsthat specifically bind to the cellular component targets remain with thecells.

In some embodiments, the methods disclosed herein can provide releasingthe plurality of nucleic acid target molecules from the sample, e.g.,cells. For example, the cells can be lysed to release the plurality ofnucleic acid target molecules. Cell lysis may be accomplished by any ofa variety of means, for example, by chemical treatment, osmotic shock,thermal treatment, mechanical treatment, optical treatment, or anycombination thereof. Cells may be lysed by addition of a cell lysisbuffer comprising a detergent (e.g., SDS, Li dodecyl sulfate, TritonX-100, Tween-20, or NP-40), an organic solvent (e.g., methanol oracetone), or digestive enzymes (e.g., proteinase K, pepsin, or trypsin),or any combination thereof.

It would be appreciated by one of ordinary skill in the art that theplurality of nucleic acid molecules can comprise a variety of nucleicacid molecules. In some embodiments, the plurality of nucleic acidmolecules can comprise, DNA molecules, RNA molecules, genomic DNAmolecules, mRNA molecules, rRNA molecules, siRNA molecules, or acombination thereof, and can be double-stranded or single-stranded. Insome embodiments, the plurality of nucleic acid molecules comprise, orcomprise about, 100, 1000, 10000, 20000, 30000, 40000, 50000, 100000,1000000, or a number or a range between any two of these values,species. In some embodiments, the plurality of nucleic acid moleculescomprises at least, or comprise at most, 100, 1000, 10000, 20000, 30000,40000, 50000, 100000, or 1000000, species. In some embodiments, theplurality of nucleic acid molecules can be from a sample, such as asingle cell, or a plurality of cells. In some embodiments, the pluralityof nucleic acid molecules can be pooled from a plurality of samples,such as a plurality of single cells.

In some embodiments, the methods disclosed herein can compriseassociating a barcode (e.g., a stochastic barcode), which can include abarcode sequence (such as a molecular label), a cell label, a samplelabel, etc., or any combination thereof, to the plurality of nucleicacid target molecules and the plurality of oligonucleotides of thecellular component binding reagents. For example, a plurality ofoligonucleotide probes comprising a stochastic barcode can be used tohybridize to the plurality of nucleic acid target molecules and theplurality of oligonucleotides of the compositions.

In some embodiments, the plurality of oligonucleotide probes can beimmobilized on solid supports. The solid supports can be free floating,e.g., beads in a solution. The solid supports can be embedded in asemi-solid or solid array. In some embodiments, the plurality ofoligonucleotide probes may not be immobilized on solid supports. Whenthe plurality of oligonucleotide probes are in close proximity to theplurality of nucleic acid target molecules and the plurality ofoligonucleotides of the cellular component binding reagents, theplurality of nucleic acid target molecules and the plurality ofoligonucleotides of the cellular component binding reagents canhybridize to the oligonucleotide probes. The oligonucleotide probes canbe contacted at a non-depletable ratio such that each distinct nucleicacid target molecules and oligonucleotides of the cellular componentbinding reagents can associate with oligonucleotide probes havingdifferent barcode sequences (e.g., molecular labels) of the disclosure.

In some embodiments, the methods disclosed herein provide detaching theoligonucleotides from the cellular component binding reagents that arespecifically bound to the cellular component targets. Detachment can beperformed in a variety of ways to separate the chemical group from thecellular component binding reagent, such as UV photocleaving, chemicaltreatment (e.g., dithiothreitol treatment), heating, enzyme treatment,or any combination thereof. Detaching the oligonucleotide from thecellular component binding reagent can be performed either before,after, or during the step of hybridizing the plurality ofoligonucleotide probes to the plurality of nucleic acid target moleculesand the plurality of oligonucleotides of the compositions.

Simultaneous Quantitative Analysis of Protein and Nucleic Acid Targets

In some embodiments, the methods disclosed herein also can be used forsimultaneous quantitative analysis of multiple types of targetmolecules, for example protein and nucleic acid targets. For example,the target molecules can be, or comprise, cellular components. FIG. 6shows a schematic illustration of an exemplary method of simultaneousquantitative analysis of both nucleic acid targets and other cellularcomponent targets (e.g., proteins) in single cells. In some embodiments,a plurality of compositions 605, 605 b, 605 c, etc., each comprising acellular component binding reagent, such as an antibody, is provided.Different cellular component binding reagents, such as antibodies, whichbind to different cellular component targets are conjugated withdifferent unique identifiers. Next, the cellular component bindingreagents can be incubated with a sample containing a plurality of cells610. The different cellular component binding reagents can specificallybind to cellular components on the cell surface, such as a cell marker,a B-cell receptor, a T-cell receptor, an antibody, a majorhistocompatibility complex, a tumor antigen, a receptor, or anycombination thereof. Unbound cellular component binding reagents can beremoved, e.g., by washing the cells with a buffer. The cells with thecellular component binding reagents can be then separated into aplurality of compartments, such as a microwell array, wherein a singlecompartment 615 is sized to fit a single cell and a single bead 620.Each bead can comprise a plurality of oligonucleotide probes, which cancomprise a cell label that is common to all oligonucleotide probes on abead, and barcode sequences (e.g., molecular label sequences). In someembodiments, each oligonucleotide probe can comprise a target bindingregion, for example, a poly(dT) sequence. The oligonucleotides 625conjugated to the cellular component binding reagent can be detachedfrom the cellular component binding reagent using chemical, optical orother means. The cell can be lysed 635 to release nucleic acids withinthe cell, such as genomic DNA or cellular mRNA 630. Cellular mRNA 630,oligonucleotides 625 or both can be captured by the oligonucleotideprobes on bead 620, for example, by hybridizing to the poly(dT)sequence. A reverse transcriptase can be used to extend theoligonucleotide probes hybridized to the cellular mRNA 630 and theoligonucleotides 625 using the cellular mRNA 630 and theoligonucleotides 625 as templates. The extension products produced bythe reverse transcriptase can be subject to amplification andsequencing. Sequencing reads can be subject to demultiplexing ofsequences or identifies of cell labels, barcodes (e.g., molecularlabels), genes, cellular component binding reagent specificoligonucleotides (e.g., antibody specific oligonucleotides), etc., whichcan give rise to a digital representation of cellular components andgene expression of each single cell in the sample.

Association of Barcodes

The oligonucleotides associated with the cellular component bindingreagents (e.g., antigen binding reagents or protein binding reagents)and/or the nucleic acid molecules may randomly associate with theoligonucleotide probes (e.g., barcodes, such as stochastic barcodes).The oligonucleotides associated with the cellular component bindingreagents, referred to herein as binding reagent oligonucleotides, canbe, or comprise oligonucleotides of the disclosure, such as an antibodyoligonucleotide, a sample indexing oligonucleotide, a cellidentification oligonucleotide, a control particle oligonucleotide, acontrol oligonucleotide, an interaction determination oligonucleotide,etc. Association can, for example, comprise hybridization of anoligonucleotide probe's target binding region to a complementary portionof the target nucleic acid molecule and/or the oligonucleotides of theprotein binding reagents. For example, a oligo(dT) region of a barcode(e.g., a stochastic barcode) can interact with a poly(A) tail of atarget nucleic acid molecule and/or a poly(A) tail of an oligonucleotideof a protein binding reagent. The assay conditions used forhybridization (e.g., buffer pH, ionic strength, temperature, etc.) canbe chosen to promote formation of specific, stable hybrids.

The disclosure provides for methods of associating a molecular labelwith a target nucleic acid and/or an oligonucleotide associated with acellular component binding reagent using reverse transcription. As areverse transcriptase can use both RNA and DNA as template. For example,the oligonucleotide originally conjugated on the cellular componentbinding reagent can be either RNA or DNA bases, or both. A bindingreagent oligonucleotide can be copied and linked (e.g., covalentlylinked) to a cell label and a barcode sequence (e.g., a molecular label)in addition to the sequence, or a portion thereof, of the bindingreagent sequence. As another example, an mRNA molecule can be copied andlinked (e.g., covalently linked) to a cell label and a barcode sequence(e.g., a molecular label) in addition to the sequence of the mRNAmolecule, or a portion thereof.

In some embodiments, molecular labels can be added by ligation of anoligonucleotide probe target binding region and a portion of the targetnucleic acid molecule and/or the oligonucleotides associated with (e.g.,currently, or previously, associated with) with cellular componentbinding reagents. For example, the target binding region may comprise anucleic acid sequence that can be capable of specific hybridization to arestriction site overhang (e.g., an EcoRI sticky-end overhang). Themethods can further comprise treating the target nucleic acids and/orthe oligonucleotides associated with cellular component binding reagentswith a restriction enzyme (e.g., EcoRI) to create a restriction siteoverhang. A ligase (e.g., T4 DNA ligase) may be used to join the twofragments.

Determining the Number or Presence of Unique Molecular Label Sequences

In some embodiments, the methods disclosed herein comprise determiningthe number or presence of unique molecular label sequences for eachunique identifier, each nucleic acid target molecule, and/or eachbinding reagent oligonucleotides (e.g., antibody oligonucleotides). Forexample, the sequencing reads can be used to determine the number ofunique molecular label sequences for each unique identifier, eachnucleic acid target molecule, and/or each binding reagentoligonucleotide. As another example, the sequencing reads can be used todetermine the presence or absence of a molecular label sequence (such asa molecular label sequence associated with a target, a binding reagentoligonucleotide, an antibody oligonucleotide, a sample indexingoligonucleotide, a cell identification oligonucleotide, a controlparticle oligonucleotide, a control oligonucleotide, an interactiondetermination oligonucleotide, etc. in the sequencing reads).

In some embodiments, the number of unique molecular label sequences foreach unique identifier, each nucleic acid target molecule, and/or eachbinding reagent oligonucleotide indicates the quantity of each cellularcomponent target (e.g., an antigen target or a protein target) and/oreach nucleic acid target molecule in the sample. In some embodiments,the quantity of a cellular component target and the quantity of itscorresponding nucleic acid target molecules, e.g., mRNA molecules, canbe compared to each other. In some embodiments, the ratio of thequantity of a cellular component target and the quantity of itscorresponding nucleic acid target molecules, e.g., mRNA molecules, canbe calculated. The cellular component targets can be, for example, cellsurface protein markers. In some embodiments, the ratio between theprotein level of a cell surface protein marker and the level of the mRNAof the cell surface protein marker is low.

The methods disclosed herein can be used for a variety of applications.For example, the methods disclosed herein can be used for proteomeand/or transcriptome analysis of a sample. In some embodiments, themethods disclosed herein can be used to identify a cellular componenttarget and/or a nucleic acid target, i.e., a biomarker, in a sample. Insome embodiments, the cellular component target and the nucleic acidtarget correspond to each other, i.e., the nucleic acid target encodesthe cellular component target. In some embodiments, the methodsdisclosed herein can be used to identify cellular component targets thathave a desired ratio between the quantity of the cellular componenttarget and the quantity of its corresponding nucleic acid targetmolecule in a sample, e.g., mRNA molecule. In some embodiments, theratio is, or is about, 0.001, 0.01, 0.1, 1, 10, 100, 1000, or a numberor a range between any two of the above values. In some embodiments, theratio is at least, or is at most, 0.001, 0.01, 0.1, 1, 10, 100, or 1000.In some embodiments, the methods disclosed herein can be used toidentify cellular component targets in a sample that the quantity of itscorresponding nucleic acid target molecule in the sample is, or isabout, 1000, 100, 10, 5, 2 1, 0, or a number or a range between any twoof these values. In some embodiments, the methods disclosed herein canbe used to identify cellular component targets in a sample that thequantity of its corresponding nucleic acid target molecule in the sampleis more than, or less than, 1000, 100, 10, 5, 2 1, or 0.

Compositions and Kits

Some embodiments disclosed herein provide kits and compositions forsimultaneous quantitative analysis of a plurality of cellular components(e.g., proteins) and/or a plurality of nucleic acid target molecules ina sample. The kits and compositions can, in some embodiments, comprise aplurality of cellular component binding reagents (e.g., a plurality ofprotein binding reagents) each conjugated with an oligonucleotide,wherein the oligonucleotide comprises a unique identifier for thecellular component binding reagent, and a plurality of oligonucleotideprobes, wherein each of the plurality of oligonucleotide probescomprises a target binding region, a barcode sequence (e.g., a molecularlabel sequence), wherein the barcode sequence is from a diverse set ofunique barcode sequences. In some embodiments, each of theoligonucleotides can comprise a molecular label, a cell label, a samplelabel, or any combination thereof. In some embodiments, each of theoligonucleotides can comprise a linker. In some embodiments, each of theoligonucleotides can comprise a binding site for an oligonucleotideprobe, such as a poly(A) tail. For example, the poly(A) tail can be,e.g., oligodA₁₈ (unanchored to a solid support) or oligoA₁₈V (anchoredto a solid support). The oligonucleotides can comprise DNA residues, RNAresidues, or both.

Disclosed herein include a plurality of sample indexing compositions.Each of the plurality of sample indexing compositions can comprise twoor more cellular component binding reagents. Each of the two or morecellular component binding reagents can be associated with a sampleindexing oligonucleotide. At least one of the two or more cellularcomponent binding reagents can be capable of specifically binding to atleast one cellular component target. The sample indexing oligonucleotidecan comprise a sample indexing sequence for identifying sample origin ofone or more cells of a sample. Sample indexing sequences of at least twosample indexing compositions of the plurality of sample indexingcompositions can comprise different sequences.

Disclosed herein include kits comprising sample indexing compositionsfor cell identification. In some embodiments. Each of two sampleindexing compositions comprises a cellular component binding reagent(e.g., a protein binding reagent) associated with a sample indexingoligonucleotide, wherein the cellular component binding reagent iscapable of specifically binding to at least one of one or more cellularcomponent targets (e.g., one or more protein targets), wherein thesample indexing oligonucleotide comprises a sample indexing sequence,and wherein sample indexing sequences of the two sample indexingcompositions comprise different sequences. In some embodiments, thesample indexing oligonucleotide comprises a molecular label sequence, abinding site for a universal primer, or a combination thereof.

Disclosed herein include kits for cell identification. In someembodiments, the kit comprises: two or more sample indexingcompositions. Each of the two or more sample indexing compositions cancomprise a cellular component binding reagent (e.g., an antigen bindingreagent) associated with a sample indexing oligonucleotide, wherein thecellular component binding reagent is capable of specifically binding toat least one of one or more cellular component targets, wherein thesample indexing oligonucleotide comprises a sample indexing sequence,and wherein sample indexing sequences of the two sample indexingcompositions comprise different sequences. In some embodiments, thesample indexing oligonucleotide comprises a molecular label sequence, abinding site for a universal primer, or a combination thereof. Disclosedherein include kits for multiplet identification. In some embodiments,the kit comprises two sample indexing compositions. Each of two sampleindexing compositions can comprise a cellular component binding reagent(e.g., an antigen binding reagent) associated with a sample indexingoligonucleotide, wherein the antigen binding reagent is capable ofspecifically binding to at least one of one or more cellular componenttargets (e.g., antigen targets), wherein the sample indexingoligonucleotide comprises a sample indexing sequence, and wherein sampleindexing sequences of the two sample indexing compositions comprisedifferent sequences.

The unique identifiers (or oligonucleotides associated with cellularcomponent binding reagents, such as binding reagent oligonucleotides,antibody oligonucleotides, sample indexing oligonucleotides, cellidentification oligonucleotides, control particle oligonucleotides,control oligonucleotides, or interaction determination oligonucleotides)can have any suitable length, for example, from about 25 nucleotides toabout 45 nucleotides long. In some embodiments, the unique identifiercan have a length that is, is about, is less than, is greater than, 4,5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90,100, 200 nucleotides, or a range that is between any two of the abovevalues.

In some embodiments, the unique identifiers are selected from a diverseset of unique identifiers. The diverse set of unique identifiers cancomprise, or comprise about, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200,300, 400, 500, 600, 700, 800, 900, 1000, 2000, 5000, or a number or arange between any two of these values, different unique identifiers. Thediverse set of unique identifiers can comprise at least, or comprise atmost, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700,800, 900, 1000, 2000, or 5000, different unique identifiers. In someembodiments, the set of unique identifiers is designed to have minimalsequence homology to the DNA or RNA sequences of the sample to beanalyzed. In some embodiments, the sequences of the set of uniqueidentifiers are different from each other, or the complement thereof,by, or by about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nucleotides, or a numberor a range between any two of these values. In some embodiments, thesequences of the set of unique identifiers are different from eachother, or the complement thereof, by at least, or by at most, 1, 2, 3,4, 5, 6, 7, 8, 9, or 10 nucleotides.

In some embodiments, the unique identifiers can comprise a binding sitefor a primer, such as universal primer. In some embodiments, the uniqueidentifiers can comprise at least two binding sites for a primer, suchas a universal primer. In some embodiments, the unique identifiers cancomprise at least three binding sites for a primer, such as a universalprimer. The primers can be used for amplification of the uniqueidentifiers, for example, by PCR amplification. In some embodiments, theprimers can be used for nested PCR reactions.

Any suitable cellular component binding reagents are contemplated inthis disclosure, such as any protein binding reagents (e.g., antibodiesor fragments thereof, aptamers, small molecules, ligands, peptides,oligonucleotides, etc., or any combination thereof). In someembodiments, the cellular component binding reagents can be polyclonalantibodies, monoclonal antibodies, recombinant antibodies, single-chainantibody (scAb), or fragments thereof, such as Fab, Fv, etc. In someembodiments, the plurality of protein binding reagents can comprise, orcomprise about, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500,600, 700, 800, 900, 1000, 2000, 5000, or a number or a range between anytwo of these values, different protein binding reagents. In someembodiments, the plurality of protein binding reagents can comprise atleast, or comprise at most, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200,300, 400, 500, 600, 700, 800, 900, 1000, 2000, or 5000, differentprotein binding reagents.

In some embodiments, the oligonucleotide is conjugated with the cellularcomponent binding reagent through a linker. In some embodiments, theoligonucleotide can be conjugated with the protein binding reagentcovalently. In some embodiment, the oligonucleotide can be conjugatedwith the protein binding reagent non-covalently. In some embodiments,the linker can comprise a chemical group that reversibly or irreversiblyattached the oligonucleotide to the protein binding reagents. Thechemical group can be conjugated to the linker, for example, through anamine group. In some embodiments, the linker can comprise a chemicalgroup that forms a stable bond with another chemical group conjugated tothe protein binding reagent. For example, the chemical group can be a UVphotocleavable group, a disulfide bond, a streptavidin, a biotin, anamine, etc. In some embodiments, the chemical group can be conjugated tothe protein binding reagent through a primary amine on an amino acid,such as lysine, or the N-terminus. The oligonucleotide can be conjugatedto any suitable site of the protein binding reagent, as long as it doesnot interfere with the specific binding between the protein bindingreagent and its protein target. In embodiments where the protein bindingreagent is an antibody, the oligonucleotide can be conjugated to theantibody anywhere other than the antigen-binding site, for example, theFc region, the C_(H)1 domain, the C_(H)2 domain, the C_(H)3 domain, theC_(L) domain, etc. In some embodiments, each protein binding reagent canbe conjugated with a single oligonucleotide molecule. In someembodiments, each protein binding reagent can be conjugated with, orwith about, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 1000, or a number or arange between any two of these values, oligonucleotide molecules,wherein each of the oligonucleotide molecule comprises the same uniqueidentifier. In some embodiments, each protein binding reagent can beconjugated with more than one oligonucleotide molecule, for example, atleast, or at most, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, or 1000,oligonucleotide molecules, wherein each of the oligonucleotide moleculecomprises the same unique identifier.

In some embodiments, the plurality of cellular component bindingreagents (e.g., protein binding reagents) are capable of specificallybinding to a plurality of cellular component targets (e.g., proteintargets) in a sample. The sample can be, or comprise, a single cell, aplurality of cells, a tissue sample, a tumor sample, a blood sample, orthe like. In some embodiments, the plurality of cellular componenttargets comprises a cell-surface protein, a cell marker, a B-cellreceptor, a T-cell receptor, an antibody, a major histocompatibilitycomplex, a tumor antigen, a receptor, or any combination thereof. Insome embodiments, the plurality of cellular component targets cancomprise intracellular proteins. In some embodiments, the plurality ofcellular component targets can be, or be about, 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%,or a number or a range between any two of these values of all cellularcomponent targets (e.g., proteins expressed or could be expressed) in anorganism. In some embodiments, the plurality of cellular componenttargets can be at least, or be at most, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%, ofall cellular component targets (e.g., proteins expressed or could beexpressed) in an organism. In some embodiments, the plurality ofcellular component targets can comprise, or comprise about, 2, 3, 4, 5,10, 20, 30, 40, 50, 100, 1000, 10000, or a number or a range between anytwo of these values, different cellular component targets. In someembodiments, the plurality of cellular component targets can comprise atleast, or comprise at most, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 1000,or 10000, different cellular component targets.

Sample Indexing Using Oligonucleotide-Conjugated Cellular ComponentBinding Reagent

Disclosed herein include methods for sample identification. In someembodiments, the method comprises: contacting one or more cells fromeach of a plurality of samples with a sample indexing composition of aplurality of sample indexing compositions, wherein each of the one ormore cells comprises one or more cellular component targets, whereineach of the plurality of sample indexing compositions comprises acellular component binding reagent associated with a sample indexingoligonucleotide, wherein the cellular component binding reagent iscapable of specifically binding to at least one of the one or morecellular component targets, wherein the sample indexing oligonucleotidecomprises a sample indexing sequence, and wherein sample indexingsequences of at least two sample indexing compositions of the pluralityof sample indexing compositions comprise different sequences; removingunbound sample indexing compositions of the plurality of sample indexingcompositions; barcoding (e.g., stochastically barcoding) the sampleindexing oligonucleotides using a plurality of barcodes (e.g.,stochastic barcodes) to create a plurality of barcoded sample indexingoligonucleotides; obtaining sequencing data of the plurality of barcodedsample indexing oligonucleotides; and identifying sample origin of atleast one cell of the one or more cells based on the sample indexingsequence of at least one barcoded sample indexing oligonucleotide of theplurality of barcoded sample indexing oligonucleotides.

In some embodiments, barcoding the sample indexing oligonucleotidesusing the plurality of barcodes comprises: contacting the plurality ofbarcodes with the sample indexing oligonucleotides to generate barcodeshybridized to the sample indexing oligonucleotides; and extending thebarcodes hybridized to the sample indexing oligonucleotides to generatethe plurality of barcoded sample indexing oligonucleotides. Extendingthe barcodes can comprise extending the barcodes using a DNA polymeraseto generate the plurality of barcoded sample indexing oligonucleotides.Extending the barcodes can comprise extending the barcodes using areverse transcriptase to generate the plurality of barcoded sampleindexing oligonucleotides.

An oligonucleotide-conjugated with an antibody, an oligonucleotide forconjugation with an antibody, or an oligonucleotide previouslyconjugated with an antibody is referred to herein as an antibodyoligonucleotide (“AbOligo”). Antibody oligonucleotides in the context ofsample indexing are referred to herein as sample indexingoligonucleotides. An antibody conjugated with an antibodyoligonucleotide is referred to herein as a hot antibody or anoligonucleotide antibody. An antibody not conjugated with an antibodyoligonucleotide is referred to herein as a cold antibody or anoligonucleotide free antibody. An oligonucleotide-conjugated with abinding reagent (e.g., a protein binding reagent), an oligonucleotidefor conjugation with a binding reagent, or an oligonucleotide previouslyconjugated with a binding reagent is referred to herein as a reagentoligonucleotide. Reagent oligonucleotides in the context of sampleindexing are referred to herein as sample indexing oligonucleotides. Abinding reagent conjugated with an antibody oligonucleotide is referredto herein as a hot binding reagent or an oligonucleotide bindingreagent. A binding reagent not conjugated with an antibodyoligonucleotide is referred to herein as a cold binding reagent or anoligonucleotide free binding reagent.

FIG. 7 shows a schematic illustration of an exemplary workflow usingoligonucleotide-associated cellular component binding reagents forsample indexing. In some embodiments, a plurality of compositions 705 a,705 b, etc., each comprising a binding reagent is provided. The bindingreagent can be a protein binding reagent, such as an antibody. Thecellular component binding reagent can comprise an antibody, a tetramer,an aptamer, a protein scaffold, or a combination thereof. The bindingreagents of the plurality of compositions 705 a, 705 b can bind to anidentical cellular component target. For example, the binding reagentsof the plurality of compositions 705, 705 b can be identical (except forthe sample indexing oligonucleotides associated with the bindingreagents).

Different compositions can include binding reagents conjugated withsample indexing oligonucleotides with different sample indexingsequences. The number of different compositions can be different indifferent implementations. In some embodiments, the number of differentcompositions can be, or be about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, or a numberor a range between any two of these values. In some embodiments, thenumber of different compositions can be at least, or be at most, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300,400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000,8000, 9000, or 10000.

In some embodiments, the sample indexing oligonucleotides of bindingreagents in one composition can include an identical sample indexingsequence. The sample indexing oligonucleotides of binding reagents inone composition may not be identical. In some embodiments, thepercentage of sample indexing oligonucleotides of binding reagents inone composition with an identical sample indexing sequence can be, or beabout, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or a number or arange between any two of these values. In some embodiments, thepercentage of sample indexing oligonucleotides of binding reagents inone composition with an identical sample indexing sequence can be atleast, or be at most, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9%.

The compositions 705 a and 705 b can be used to label samples ofdifferent samples. For example, the sample indexing oligonucleotides ofthe cellular component binding reagent in the composition 705 a can haveone sample indexing sequence and can be used to label cells 710 a, shownas black circles, in a sample 707 a, such as a sample of a patient. Thesample indexing oligonucleotides of the cellular component bindingreagents in the composition 705 b can have another sample indexingsequence and can be used to label cells 710 b, shown as hatched circles,in a sample 707 b, such as a sample of another patient or another sampleof the same patient. The cellular component binding reagents canspecifically bind to cellular component targets or proteins on the cellsurface, such as a cell marker, a B-cell receptor, a T-cell receptor, anantibody, a major histocompatibility complex, a tumor antigen, areceptor, or any combination thereof. Unbound cellular component bindingreagents can be removed, e.g., by washing the cells with a buffer.

The cells with the cellular component binding reagents can be thenseparated into a plurality of compartments, such as a microwell array,wherein a single compartment 715 a, 715 b is sized to fit a single cell710 a and a single bead 720 a or a single cell 710 b and a single bead720 b. Each bead 720 a, 720 b can comprise a plurality ofoligonucleotide probes, which can comprise a cell label that is commonto all oligonucleotide probes on a bead, and molecular label sequences.In some embodiments, each oligonucleotide probe can comprise a targetbinding region, for example, a poly(dT) sequence. The sample indexingoligonucleotides 725 a conjugated to the cellular component bindingreagent of the composition 705 a can be configured to be (or can be)detachable or non-detachable from the cellular component bindingreagent. The sample indexing oligonucleotides 725 a conjugated to thecellular component binding reagent of the composition 705 a can bedetached from the cellular component binding reagent using chemical,optical or other means. The sample indexing oligonucleotides 725 bconjugated to the cellular component binding reagent of the composition705 b can be configured to be (or can be) detachable or non-detachablefrom the cellular component binding reagent. The sample indexingoligonucleotides 725 b conjugated to the cellular component bindingreagent of the composition 705 b can be detached from the cellularcomponent binding reagent using chemical, optical or other means.

The cell 710 a can be lysed to release nucleic acids within the cell 710a, such as genomic DNA or cellular mRNA 730 a. The lysed cell 735 a isshown as a dotted circle. Cellular mRNA 730 a, sample indexingoligonucleotides 725 a, or both can be captured by the oligonucleotideprobes on bead 720 a, for example, by hybridizing to the poly(dT)sequence. A reverse transcriptase can be used to extend theoligonucleotide probes hybridized to the cellular mRNA 730 a and theoligonucleotides 725 a using the cellular mRNA 730 a and theoligonucleotides 725 a as templates. The extension products produced bythe reverse transcriptase can be subject to amplification andsequencing.

Similarly, the cell 710 b can be lysed to release nucleic acids withinthe cell 710 b, such as genomic DNA or cellular mRNA 730 b. The lysedcell 735 b is shown as a dotted circle. Cellular mRNA 730 b, sampleindexing oligonucleotides 725 b, or both can be captured by theoligonucleotide probes on bead 720 b, for example, by hybridizing to thepoly(dT) sequence. A reverse transcriptase can be used to extend theoligonucleotide probes hybridized to the cellular mRNA 730 b and theoligonucleotides 725 b using the cellular mRNA 730 b and theoligonucleotides 725 b as templates. The extension products produced bythe reverse transcriptase can be subject to amplification andsequencing.

Sequencing reads can be subject to demultiplexing of cell labels,molecular labels, gene identities, and sample identities (e.g., in termsof sample indexing sequences of sample indexing oligonucleotides 725 aand 725 b). Demultiplexing of cell labels, molecular labels, and geneidentities can give rise to a digital representation of gene expressionof each single cell in the sample. Demultiplexing of cell labels,molecular labels, and sample identities, using sample indexing sequencesof sample indexing oligonucleotides, can be used to determine a sampleorigin.

In some embodiments, cellular component binding reagents againstcellular component binding reagents on the cell surface can beconjugated to a library of unique sample indexing oligonucleotides toallow cells to retain sample identity. For example, antibodies againstcell surface markers can be conjugated to a library of unique sampleindexing oligonucleotides to allow cells to retain sample identity. Thiswill enable multiple samples to be loaded onto the same Rhapsody™cartridge as information pertaining sample source is retained throughoutlibrary preparation and sequencing. Sample indexing can allow multiplesamples to be run together in a single experiment, simplifying andshortening experiment time, and eliminating batch effect.

Disclosed herein include methods for sample identification. In someembodiments, the method comprise: contacting one or more cells from eachof a plurality of samples with a sample indexing composition of aplurality of sample indexing compositions, wherein each of the one ormore cells comprises one or more cellular component targets, whereineach of the plurality of sample indexing compositions comprises acellular component binding reagent associated with a sample indexingoligonucleotide, wherein the cellular component binding reagent iscapable of specifically binding to at least one of the one or morecellular component targets, wherein the sample indexing oligonucleotidecomprises a sample indexing sequence, and wherein sample indexingsequences of at least two sample indexing compositions of the pluralityof sample indexing compositions comprise different sequences; removingunbound sample indexing compositions of the plurality of sample indexingcompositions. The method can include barcoding (e.g., stochasticallybarcoding) the sample indexing oligonucleotides using a plurality ofbarcodes (e.g., stochastic barcodes) to create a plurality of barcodedsample indexing oligonucleotides; obtaining sequencing data of theplurality of barcoded sample indexing oligonucleotides; and identifyingsample origin of at least one cell of the one or more cells based on thesample indexing sequence of at least one barcoded sample indexingoligonucleotide of the plurality of barcoded sample indexingoligonucleotides.

In some embodiments, the method for sample identification comprises:contacting one or more cells from each of a plurality of samples with asample indexing composition of a plurality of sample indexingcompositions, wherein each of the one or more cells comprises one ormore cellular component targets, wherein each of the plurality of sampleindexing compositions comprises a cellular component binding reagentassociated with a sample indexing oligonucleotide, wherein the cellularcomponent binding reagent is capable of specifically binding to at leastone of the one or more cellular component targets, wherein the sampleindexing oligonucleotide comprises a sample indexing sequence, andwherein sample indexing sequences of at least two sample indexingcompositions of the plurality of sample indexing compositions comprisedifferent sequences; removing unbound sample indexing compositions ofthe plurality of sample indexing compositions; and identifying sampleorigin of at least one cell of the one or more cells based on the sampleindexing sequence of at least one sample indexing oligonucleotide of theplurality of sample indexing compositions.

In some embodiments, identifying the sample origin of the at least onecell comprises: barcoding (e.g., stochastically barcoding) sampleindexing oligonucleotides of the plurality of sample indexingcompositions using a plurality of barcodes (e.g., stochastic barcodes)to create a plurality of barcoded sample indexing oligonucleotides;obtaining sequencing data of the plurality of barcoded sample indexingoligonucleotides; and identifying the sample origin of the cell based onthe sample indexing sequence of at least one barcoded sample indexingoligonucleotide of the plurality of barcoded sample indexingoligonucleotides. In some embodiments, barcoding the sample indexingoligonucleotides using the plurality of barcodes to create the pluralityof barcoded sample indexing oligonucleotides comprises stochasticallybarcoding the sample indexing oligonucleotides using a plurality ofstochastic barcodes to create a plurality of stochastically barcodedsample indexing oligonucleotides.

In some embodiments, identifying the sample origin of the at least onecell can comprise identifying the presence or absence of the sampleindexing sequence of at least one sample indexing oligonucleotide of theplurality of sample indexing compositions. Identifying the presence orabsence of the sample indexing sequence can comprise: replicating the atleast one sample indexing oligonucleotide to generate a plurality ofreplicated sample indexing oligonucleotides; obtaining sequencing dataof the plurality of replicated sample indexing oligonucleotides; andidentifying the sample origin of the cell based on the sample indexingsequence of a replicated sample indexing oligonucleotide of theplurality of sample indexing oligonucleotides that correspond to theleast one barcoded sample indexing oligonucleotide in the sequencingdata.

In some embodiments, replicating the at least one sample indexingoligonucleotide to generate the plurality of replicated sample indexingoligonucleotides comprises: prior to replicating the at least onebarcoded sample indexing oligonucleotide, ligating a replicating adaptorto the at least one barcoded sample indexing oligonucleotide.Replicating the at least one barcoded sample indexing oligonucleotidecan comprise replicating the at least one barcoded sample indexingoligonucleotide using the replicating adaptor ligated to the at leastone barcoded sample indexing oligonucleotide to generate the pluralityof replicated sample indexing oligonucleotides.

In some embodiments, replicating the at least one sample indexingoligonucleotide to generate the plurality of replicated sample indexingoligonucleotides comprises: prior to replicating the at least onebarcoded sample indexing oligonucleotide, contacting a capture probewith the at least one sample indexing oligonucleotide to generate acapture probe hybridized to the sample indexing oligonucleotide; andextending the capture probe hybridized to the sample indexingoligonucleotide to generate a sample indexing oligonucleotide associatedwith the capture probe. Replicating the at least one sample indexingoligonucleotide can comprise replicating the sample indexingoligonucleotide associated with the capture probe to generate theplurality of replicated sample indexing oligonucleotides.

Cell Overloading and Multiplet Identification

Also disclosed herein include methods, kits and systems for identifyingcell overloading and multiplet. Such methods, kits and systems can beused in, or in combination with, any suitable methods, kits and systemsdisclosed herein, for example the methods, kits and systems formeasuring cellular component expression level (such as proteinexpression level) using cellular component binding reagents associatedwith oligonucleotides.

Using current cell-loading technology, when about 20000 cells are loadedinto a microwell cartridge or array with ˜60000 microwells, the numberof microwells or droplets with two or more cells (referred to asdoublets or multiplets) can be minimal. However, when the number ofcells loaded increases, the number of microwells or droplets withmultiple cells can increase significantly. For example, when about 50000cells are loaded into about 60000 microwells of a microwell cartridge orarray, the percentage of microwells with multiple cells can be quitehigh, such as 11-14%. Such loading of high number of cells intomicrowells can be referred to as cell overloading. However, if the cellsare divided into a number of groups (e.g., 5), and cells in each groupare labeled with sample indexing oligonucleotides with distinct sampleindexing sequences, a cell label (e.g., a cell label of a barcode, suchas a stochastic barcode) associated with two or more sample indexingsequences can be identified in sequencing data and removed fromsubsequent processing. In some embodiments, the cells are divided into alarge number of groups (e.g., 10000), and cells in each group arelabeled with sample indexing oligonucleotides with distinct sampleindexing sequences, a sample label associated with two or more sampleindexing sequences can be identified in sequencing data and removed fromsubsequent processing. In some embodiments, different cells are labeledwith cell identification oligonucleotides with distinct cellidentification sequences, a cell identification sequence associated withtwo or more cell identification oligonucleotides can be identified insequencing data and removed from subsequent processing. Such highernumber of cells can be loaded into microwells relative to the number ofmicrowells in a microwell cartridge or array.

Disclosed herein include methods for sample identification. In someembodiments, the method comprises: contacting a first plurality of cellsand a second plurality of cells with two sample indexing compositionsrespectively, wherein each of the first plurality of cells and each ofthe second plurality of cells comprise one or more cellular components,wherein each of the two sample indexing compositions comprises acellular component binding reagent associated with a sample indexingoligonucleotide, wherein the cellular component binding reagent iscapable of specifically binding to at least one of the one or morecellular components, wherein the sample indexing oligonucleotidecomprises a sample indexing sequence, and wherein sample indexingsequences of the two sample indexing compositions comprise differentsequences; barcoding the sample indexing oligonucleotides using aplurality of barcodes to create a plurality of barcoded sample indexingoligonucleotides, wherein each of the plurality of barcodes comprises acell label sequence, a barcode sequence (e.g., a molecular labelsequence), and a target-binding region, wherein the barcode sequences ofat least two barcodes of the plurality of barcodes comprise differentsequences, and wherein at least two barcodes of the plurality ofbarcodes comprise an identical cell label sequence; obtaining sequencingdata of the plurality of barcoded sample indexing oligonucleotides; andidentifying one or more cell label sequences that is each associatedwith two or more sample indexing sequences in the sequencing dataobtained; and removing the sequencing data associated with the one ormore cell label sequences that is each associated with two or moresample indexing sequences from the sequencing data obtained and/orexcluding the sequencing data associated with the one or more cell labelsequences that is each associated with two or more sample indexingsequences from subsequent analysis (e.g., single cell mRNA profiling, orwhole transcriptome analysis). In some embodiments, the sample indexingoligonucleotide comprises a barcode sequence (e.g., a molecular labelsequence), a binding site for a universal primer, or a combinationthereof.

For example, the method can be used to load 50000 or more cells(compared to 10000-20000 cells) using sample indexing. Sample indexingcan use oligonucleotide-conjugated cellular component binding reagents(e.g., antibodies) or cellular component binding reagents against acellular component (e.g., a universal protein marker) to label cellsfrom different samples with a unique sample index. When two or morecells from different samples, two or more cells from differentpopulations of cells of a sample, or two or more cells of a sample, arecaptured in the same microwell or droplet, the combined “cell” (orcontents of the two or more cells) can be associated with sampleindexing oligonucleotides with different sample indexing sequences (orcell identification oligonucleotides with different cell identificationsequences). The number of different populations of cells can bedifferent in different implementations. In some embodiments, the numberof different populations can be, or be about, 2, 3, 4, 5, 6, 7, 8, 9,10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or a number or a range betweenany two of these values. In some embodiments, the number of differentpopulations can be at least, or be at most, 2, 3, 4, 5, 6, 7, 8, 9, 10,20, 30, 40, 50, 60, 70, 80, 90, or 100. The number, or the averagenumber, of cells in each population can be different in differentimplementations. In some embodiments, the number, or the average number,of cells in each population can be, or be about, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or a number or a rangebetween any two of these values. In some embodiments, the number, or theaverage number, of cells in each population can be at least, or be atmost, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or100. When the number, or the average number, of cells in each populationis sufficiently small (e.g., equal to, or fewer than, 50, 25, 10, 5, 4,3, 2, or 1 cells per population), the sample indexing composition forcell overloading and multiplet identification can be referred to as cellidentification compositions.

Cells of a sample can be divided into multiple populations by aliquotingthe cells of the sample into the multiple populations. A “cell”associated with more than one sample indexing sequence in the sequencingdata can be identified as a “multiplet” based on two or more sampleindexing sequences associated with one cell label sequence (e.g., a celllabel sequence of a barcode, such as a stochastic barcode) in thesequencing data. The sequencing data of a combined “cell” is alsoreferred to herein as a multiplet. A multiplet can be a doublet, atriplet, a quartet, a quintet, a sextet, a septet, an octet, a nonet, orany combination thereof. A multiplet can be any n-plet. In someembodiments, n is, or is about, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, or a range between any two of these values.In some embodiments, n is at least, or is at most, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

When determining expression profiles of single cells, two cells may beidentified as one cell and the expression profiles of the two cells maybe identified as the expression profile for one cell (referred to as adoublet expression profile). For example, when determining expressionprofiles of two cells using barcoding (e.g., stochastic barcoding), themRNA molecules of the two cells may be associated with barcodes havingthe same cell label. As another example, two cells may be associatedwith one particle (e.g., a bead). The particle can include barcodes withthe same cell label. After lysing the cells, the mRNA molecules in thetwo cells can be associated with the barcodes of the particle, thus thesame cell label. Doublet expression profiles can skew the interpretationof the expression profiles.

A doublet can refer to a combined “cell” associated with two sampleindexing oligonucleotides with different sample indexing sequences. Adoublet can also refer to a combined “cell” associated with sampleindexing oligonucleotides with two sample indexing sequences. A doubletcan occur when two cells associated with two sample indexingoligonucleotides of different sequences (or two or more cells associatedwith sample indexing oligonucleotides with two different sample indexingsequences) are captured in the same microwell or droplet, the combined“cell” can be associated with two sample indexing oligonucleotides withdifferent sample indexing sequences. A triplet can refer to a combined“cell” associated with three sample indexing oligonucleotides all withdifferent sample indexing sequences, or a combined “cell” associatedwith sample indexing oligonucleotides with three different sampleindexing sequences. A quartet can refer to a combined “cell” associatedwith four sample indexing oligonucleotides all with different sampleindexing sequences, or a combined “cell” associated with sample indexingoligonucleotides with four different sample indexing sequences. Aquintet can refer to a combined “cell” associated with five sampleindexing oligonucleotides all with different sample indexing sequences,or a combined “cell” associated with sample indexing oligonucleotideswith five different sample indexing sequences. A sextet can refer to acombined “cell” associated with six sample indexing oligonucleotides allwith different sample indexing sequences, or a combined “cell”associated with sample indexing oligonucleotides with six differentsample indexing sequences. A septet can refer to a combined “cell”associated with seven sample indexing oligonucleotides all withdifferent sample indexing sequences, or a combined “cell” associatedwith sample indexing oligonucleotides with seven different sampleindexing sequences. A octet can refer to a combined “cell” associatedwith eight sample indexing oligonucleotides all with different sampleindexing sequences, or a combined “cell” associated with sample indexingoligonucleotides with eight different sample indexing sequences. A nonetcan refer to a combined “cell” associated with nine sample indexingoligonucleotides all with different sample indexing sequences, or acombined “cell” associated with sample indexing oligonucleotides withnine different sample indexing sequences. A multiplet can occur when twoor more cells associated with two or more sample indexingoligonucleotides of different sequences (or two or more cells associatedwith sample indexing oligonucleotides with two or more different sampleindexing sequences) are captured in the same microwell or droplet, thecombined “cell” can be associated with sample indexing oligonucleotideswith two or more different sample indexing sequences.

As another example, the method can be used for multiplet identification,whether in the context of sample overloading or in the context ofloading cells onto microwells of a microwell array or generatingdroplets containing cells. When two or more cells are loaded into onemicrowell, the resulting data from the combined “cell” (or contents ofthe two or more cells) is a multiplet with aberrant gene expressionprofile. By using sample indexing, one can recognize some of thesemultiplets by looking for cell labels that are each associated with orassigned to two or more sample indexing oligonucleotides with differentsample indexing sequences (or sample indexing oligonucleotides with twoor more sample indexing sequences). With sample indexing sequence, themethods disclosed herein can be used for multiplet identification(whether in the context of sample overloading or not, or in the contextof loading cells onto microwells of a microwell array or generatingdroplets containing cells). In some embodiments, the method comprises:contacting a first plurality of cells and a second plurality of cellswith two sample indexing compositions respectively, wherein each of thefirst plurality of cells and each of the second plurality of cellscomprise one or more cellular components, wherein each of the two sampleindexing compositions comprises a cellular component binding reagentassociated with a sample indexing oligonucleotide, wherein the cellularcomponent binding reagent is capable of specifically binding to at leastone of the one or more cellular components, wherein the sample indexingoligonucleotide comprises a sample indexing sequence, and wherein sampleindexing sequences of the two sample indexing compositions comprisedifferent sequences; barcoding the sample indexing oligonucleotidesusing a plurality of barcodes to create a plurality of barcoded sampleindexing oligonucleotides, wherein each of the plurality of barcodescomprises a cell label sequence, a barcode sequence (e.g., a molecularlabel sequence), and a target-binding region, wherein barcode sequencesof at least two barcodes of the plurality of barcodes comprise differentsequences, and wherein at least two barcodes of the plurality ofbarcodes comprise an identical cell label sequence; obtaining sequencingdata of the plurality of barcoded sample indexing oligonucleotides; andidentifying one or more multiplet cell label sequences that is eachassociated with two or more sample indexing sequences in the sequencingdata obtained.

The number of cells that can be loaded onto microwells of a microwellcartridge or into droplets generated using a microfluidics device can belimited by the multiplet rate. Loading more cells can result in moremultiplets, which can be hard to identify and create noise in the singlecell data. With sample indexing, the method can be used to moreaccurately label or identify multiplets and remove the multiplets fromthe sequencing data or subsequent analysis. Being able to identifymultiplets with higher confidence can increase user tolerance for themultiplet rate and load more cells onto each microwell cartridge orgenerating droplets with at least one cell each.

In some embodiments, contacting the first plurality of cells and thesecond plurality of cells with the two sample indexing compositionsrespectively comprises: contacting the first plurality of cells with afirst sample indexing compositions of the two sample indexingcompositions; and contacting the first plurality of cells with a secondsample indexing compositions of the two sample indexing compositions.The number of pluralities of cells and the number of pluralities ofsample indexing compositions can be different in differentimplementations. In some embodiments, the number of pluralities of cellsand/or sample indexing compositions can be, or be about, 2, 3, 4, 5, 6,7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500,600, 700, 800, 900, 1000, 10000, 100000, 1000000, or a number or a rangebetween any two of these values. In some embodiments, the number ofpluralities of cells and/or sample indexing compositions can be atleast, or be at most, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60,70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 10000,100000, or 1000000. The number of cells can be different in differentimplementations. In some embodiments, the number, or the average number,of cells can be, or be about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40,50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000,10000, 100000, 1000000, or a number or a range between any two of thesevalues. In some embodiments, the number, or the average number, or cellscan be at least, or be at most, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40,50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000,10000, 100000, or 1000000.

In some embodiments, the method comprises: removing unbound sampleindexing compositions of the two sample indexing compositions. Removingthe unbound sample indexing compositions can comprise washing cells ofthe first plurality of cells and the second plurality of cells with awashing buffer. Removing the unbound sample indexing compositions cancomprise selecting cells bound to at least one cellular componentbinding reagent of the two sample indexing compositions using flowcytometry. In some embodiments, the method comprises: lysing the one ormore cells from each of the plurality of samples.

In some embodiments, the sample indexing oligonucleotide is configuredto be (or can be) detachable or non-detachable from the cellularcomponent binding reagent. The method can comprise detaching the sampleindexing oligonucleotide from the cellular component binding reagent.Detaching the sample indexing oligonucleotide can comprise detaching thesample indexing oligonucleotide from the cellular component bindingreagent by UV photocleaving, chemical treatment (e.g., using reducingreagent, such as dithiothreitol), heating, enzyme treatment, or anycombination thereof.

In some embodiments, barcoding the sample indexing oligonucleotidesusing the plurality of barcodes comprises: contacting the plurality ofbarcodes with the sample indexing oligonucleotides to generate barcodeshybridized to the sample indexing oligonucleotides; and extending thebarcodes hybridized to the sample indexing oligonucleotides to generatethe plurality of barcoded sample indexing oligonucleotides. Extendingthe barcodes can comprise extending the barcodes using a DNA polymeraseto generate the plurality of barcoded sample indexing oligonucleotides.Extending the barcodes can comprise extending the barcodes using areverse transcriptase to generate the plurality of barcoded sampleindexing oligonucleotides.

In some embodiments, the method comprises: amplifying the plurality ofbarcoded sample indexing oligonucleotides to produce a plurality ofamplicons. Amplifying the plurality of barcoded sample indexingoligonucleotides can comprise amplifying, using polymerase chainreaction (PCR), at least a portion of barcode sequence (e.g., themolecular label sequence) and at least a portion of the sample indexingoligonucleotide. In some embodiments, obtaining the sequencing data ofthe plurality of barcoded sample indexing oligonucleotides can compriseobtaining sequencing data of the plurality of amplicons. Obtaining thesequencing data comprises sequencing at least a portion of the barcodesequence and at least a portion of the sample indexing oligonucleotide.In some embodiments, identifying the sample origin of the at least onecell comprises identifying sample origin of the plurality of barcodedtargets based on the sample indexing sequence of the at least onebarcoded sample indexing oligonucleotide.

In some embodiments, barcoding the sample indexing oligonucleotidesusing the plurality of barcodes to create the plurality of barcodedsample indexing oligonucleotides comprises stochastically barcoding thesample indexing oligonucleotides using a plurality of stochasticbarcodes to create a plurality of stochastically barcoded sampleindexing oligonucleotides.

In some embodiments, the method includes: barcoding a plurality oftargets of the cell using the plurality of barcodes to create aplurality of barcoded targets, wherein each of the plurality of barcodescomprises a cell label sequence, and wherein at least two barcodes ofthe plurality of barcodes comprise an identical cell label sequence; andobtaining sequencing data of the barcoded targets. Barcoding theplurality of targets using the plurality of barcodes to create theplurality of barcoded targets can include: contacting copies of thetargets with target-binding regions of the barcodes; and reversetranscribing the plurality targets using the plurality of barcodes tocreate a plurality of reverse transcribed targets.

In some embodiments, the method comprises: prior to obtaining thesequencing data of the plurality of barcoded targets, amplifying thebarcoded targets to create a plurality of amplified barcoded targets.Amplifying the barcoded targets to generate the plurality of amplifiedbarcoded targets can comprise: amplifying the barcoded targets bypolymerase chain reaction (PCR). Barcoding the plurality of targets ofthe cell using the plurality of barcodes to create the plurality ofbarcoded targets can comprise stochastically barcoding the plurality oftargets of the cell using a plurality of stochastic barcodes to create aplurality of stochastically barcoded targets.

In some embodiments, the method for cell identification comprise:contacting a first plurality of one or more cells and a second pluralityof one or more cells with two cell identification compositionsrespectively, wherein each of the first plurality of one or more cellsand each of the second plurality of one or more cells comprise one ormore cellular components, wherein each of the two cell identificationcompositions comprises a cellular component binding reagent associatedwith a cell identification oligonucleotide, wherein the cellularcomponent binding reagent is capable of specifically binding to at leastone of the one or more cellular components, wherein the cellidentification oligonucleotide comprises a cell identification sequence,and wherein cell identification sequences of the two cell identificationcompositions comprise different sequences; barcoding the cellidentification oligonucleotides using a plurality of barcodes to createa plurality of barcoded cell identification oligonucleotides, whereineach of the plurality of barcodes comprises a cell label sequence, abarcode sequence (e.g., a molecular label sequence), and atarget-binding region, wherein the barcode sequences of at least twobarcodes of the plurality of barcodes comprise different sequences, andwherein at least two barcodes of the plurality of barcodes comprise anidentical cell label sequence; obtaining sequencing data of theplurality of barcoded cell identification oligonucleotides; andidentifying one or more cell label sequences that is each associatedwith two or more cell identification sequences in the sequencing dataobtained; and removing the sequencing data associated with the one ormore cell label sequences that is each associated with two or more cellidentification sequences from the sequencing data obtained and/orexcluding the sequencing data associated with the one or more cell labelsequences that is each associated with two or more cell identificationsequences from subsequent analysis (e.g., single cell mRNA profiling, orwhole transcriptome analysis). In some embodiments, the cellidentification oligonucleotide comprises a barcode sequence (e.g., amolecular label sequence), a binding site for a universal primer, or acombination thereof.

A multiplet (e.g., a doublet, triplet, etc.) can occur when two or morecells associated with two or more cell identification oligonucleotidesof different sequences (or two or more cells associated with cellidentification oligonucleotides with two or more different cellidentification sequences) are captured in the same microwell or droplet,the combined “cell” can be associated with cell identificationoligonucleotides with two or more different cell identificationsequences.

Cell identification compositions can be used for multipletidentification, whether in the context of cell overloading or in thecontext of loading cells onto microwells of a microwell array orgenerating droplets containing cells. When two or more cells are loadedinto one microwell, the resulting data from the combined “cell” (orcontents of the two or more cells) is a multiplet with aberrant geneexpression profile. By using cell identification, one can recognize someof these multiplets by looking for cell labels (e.g., cell labels ofbarcodes, such as stochastic barcodes) that are each associated with orassigned to two or more cell identification oligonucleotides withdifferent cell identification sequences (or cell identificationoligonucleotides with two or more cell identification sequences). Withcell identification sequence, the methods disclosed herein can be usedfor multiplet identification (whether in the context of sampleoverloading or not, or in the context of loading cells onto microwellsof a microwell array or generating droplets containing cells). In someembodiments, the method comprises: contacting a first plurality of oneor more cells and a second plurality of one or more cells with two cellidentification compositions respectively, wherein each of the firstplurality of one or more cells and each of the second plurality of oneor more cells comprise one or more cellular components, wherein each ofthe two cell identification compositions comprises a cellular componentbinding reagent associated with a cell identification oligonucleotide,wherein the cellular component binding reagent is capable ofspecifically binding to at least one of the one or more cellularcomponents, wherein the cell identification oligonucleotide comprises acell identification sequence, and wherein cell identification sequencesof the two cell identification compositions comprise differentsequences; barcoding the cell identification oligonucleotides using aplurality of barcodes to create a plurality of barcoded cellidentification oligonucleotides, wherein each of the plurality ofbarcodes comprises a cell label sequence, a barcode sequence (e.g., amolecular label sequence), and a target-binding region, wherein barcodesequences of at least two barcodes of the plurality of barcodes comprisedifferent sequences, and wherein at least two barcodes of the pluralityof barcodes comprise an identical cell label sequence; obtainingsequencing data of the plurality of barcoded cell identificationoligonucleotides; and identifying one or more multiplet cell labelsequences that is each associated with two or more cell identificationsequences in the sequencing data obtained.

The number of cells that can be loaded onto microwells of a microwellcartridge or into droplets generated using a microfluidics device can belimited by the multiplet rate. Loading more cells can result in moremultiplets, which can be hard to identify and create noise in the singlecell data. With cell identification, the method can be used to moreaccurately label or identify multiplets and remove the multiplets fromthe sequencing data or subsequent analysis. Being able to identifymultiplets with higher confidence can increase user tolerance for themultiplet rate and load more cells onto each microwell cartridge orgenerating droplets with at least one cell each.

In some embodiments, contacting the first plurality of one or more cellsand the second plurality of one or more cells with the two cellidentification compositions respectively comprises: contacting the firstplurality of one or more cells with a first cell identificationcompositions of the two cell identification compositions; and contactingthe first plurality of one or more cells with a second cellidentification compositions of the two cell identification compositions.The number of pluralities of cell identification compositions can bedifferent in different implementations. In some embodiments, the numberof cell identification compositions can be, or be about, 2, 3, 4, 5, 6,7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500,600, 700, 800, 900, 1000, 10000, 100000, 1000000, or a number or a rangebetween any two of these values. In some embodiments, the number of cellidentification compositions can be at least, or be at most, 2, 3, 4, 5,6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500,600, 700, 800, 900, 1000, 10000, 100000, or 1000000. The number, oraverage number, of cells in each plurality of one or more cells can bedifferent in different implementations. In some embodiments, the number,or average number, of cells in each plurality of one or more cells canbe, or be about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70,80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 10000,100000, 1000000, or a number or a range between any two of these values.In some embodiments, the number, or average number, of cells in eachplurality of one or more cells can be at least, or be at most, 2, 3, 4,5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400,500, 600, 700, 800, 900, 1000, 10000, 100000, or 1000000.

In some embodiments, the method comprises removing unbound cellidentification compositions of the two cell identification compositions.Removing the unbound cell identification compositions can comprisewashing cells of the first plurality of one or more cells and the secondplurality of one or more cells with a washing buffer. Removing theunbound cell identification compositions can comprise selecting cellsbound to at least one cellular component binding reagent of the two cellidentification compositions using flow cytometry. In some embodiments,the method comprises lysing the one or more cells from each of theplurality of samples.

In some embodiments, the cell identification oligonucleotide isconfigured to be (or can be) detachable or non-detachable from thecellular component binding reagent. The method can comprise detachingthe cell identification oligonucleotide from the cellular componentbinding reagent. Detaching the cell identification oligonucleotide cancomprise detaching the cell identification oligonucleotide from thecellular component binding reagent by UV photocleaving, chemicaltreatment (e.g., using reducing reagent, such as dithiothreitol),heating, enzyme treatment, or any combination thereof.

In some embodiments, barcoding the cell identification oligonucleotidesusing the plurality of barcodes comprises: contacting the plurality ofbarcodes with the cell identification oligonucleotides to generatebarcodes hybridized to the cell identification oligonucleotides; andextending the barcodes hybridized to the cell identificationoligonucleotides to generate the plurality of barcoded cellidentification oligonucleotides. Extending the barcodes can compriseextending the barcodes using a DNA polymerase to generate the pluralityof barcoded cell identification oligonucleotides. Extending the barcodescan comprise extending the barcodes using a reverse transcriptase togenerate the plurality of barcoded cell identification oligonucleotides.

In some embodiments, the method comprises: amplifying the plurality ofbarcoded cell identification oligonucleotides to produce a plurality ofamplicons. Amplifying the plurality of barcoded cell identificationoligonucleotides can comprise amplifying, using polymerase chainreaction (PCR), at least a portion of barcode sequence (e.g., themolecular label sequence) and at least a portion of the cellidentification oligonucleotide. In some embodiments, obtaining thesequencing data of the plurality of barcoded cell identificationoligonucleotides can comprise obtaining sequencing data of the pluralityof amplicons. Obtaining the sequencing data comprises sequencing atleast a portion of the barcode sequence and at least a portion of thecell identification oligonucleotide. In some embodiments, identifyingthe sample origin of the at least one cell comprises identifying sampleorigin of the plurality of barcoded targets based on the cellidentification sequence of the at least one barcoded cell identificationoligonucleotide.

In some embodiments, barcoding the cell identification oligonucleotidesusing the plurality of barcodes to create the plurality of barcoded cellidentification oligonucleotides comprises stochastically barcoding thecell identification oligonucleotides using a plurality of stochasticbarcodes to create a plurality of stochastically barcoded cellidentification oligonucleotides.

Oligonucleotide-Conjugated Antibodies

Unique Molecular Label Sequence

In some embodiments, the oligonucleotide associated with a cellularcomponent-binding reagent (e.g., antibody oligonucleotide (“AbOligo” or“AbO”), binding reagent oligonucleotide, cellular component-bindingreagent specific oligonucleotides, sample indexing oligonucleotides)comprises a unique molecular label sequence (also referred to as amolecular index (MI), “molecular barcode,” or Unique MolecularIdentifier (UMI)). In some embodiments, binding reagent oligonucleotidespecies comprising molecule barcodes as described herein reduce bias byincreasing sensitivity, decreasing relative standard error, orincreasing sensitivity and/or reducing standard error. The moleculebarcode can comprise a unique sequence, so that when multiple samplenucleic acids (which can be the same and/or different from each other)are associated one-to-one with molecule barcodes, different samplenucleic acids can be differentiated from each other by the moleculebarcodes. As such, even if a sample comprises two nucleic acids havingthe same sequence, each of these two nucleic acids can be labeled with adifferent molecule barcode, so that nucleic acids in the population canbe quantified, even after amplification. The molecule barcode cancomprise a nucleic acid sequence of at least 5 nucleotides, for exampleat least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides, includingranges between any two of the listed values, for example 5-50, 5-45,5-40, 5-35, 5-30, 5-25, 5-20, 5-15, 5-14, 5-13, 5-12, 5-11, 5-10, 5-9,5-8, 5-7, 5-6, 6-50, 6-45, 6-40, 6-35, 6-30, 6-25, 6-20, 6-15, 6-14,6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 6-7, 7-50, 7-45, 7-40, 7-35, 7-30,7-25, 7-20, 7-15, 7-14, 7-13, 7-12, 7-11, 7-10, 7-9, 7-8, 8-50, 8-45,8-40, 8-35, 8-30, 8-25, 8-20, 8-15, 8-14, 8-13, 8-12, 8-11, 8-10, 8-9,9-50, 9-45, 9-40, 9-35, 9-30, 9-25, 9-20, 9-15, 9-14, 9-13, 9-12, 9-11,9-10, 10-50, 10-45, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 10-14,10-13, 10-12, or 10-11 nucleotides. In some embodiments, the nucleicacid sequence of the molecule barcode comprises a unique sequence, forexample, so that each unique oligonucleotide species in a compositioncomprises a different molecule barcode. In some embodiments, two or moreunique oligonucleotide species can comprise the same molecule barcode,but still differ from each other. For example, if the uniqueoligonucleotide species include sample barcodes, each uniqueoligonucleotide species with a particular sample barcode can comprise adifferent molecule barcode. In some embodiments, a compositioncomprising unique oligonucleotide species comprises a molecule barcodediversity of at least 1000 different molecule barcodes, and thus atleast 1000 unique oligonucleotide species. In some embodiments, acomposition comprising unique oligonucleotide species comprises amolecule barcode diversity of at least 6,500 different moleculebarcodes, and thus at least 6,500 unique oligonucleotide species. Insome embodiments, a composition comprising unique oligonucleotidespecies comprises a molecule barcode diversity of at least 65,000different molecule barcodes, and thus at least 65,000 uniqueoligonucleotide species.

In some embodiments, the unique molecular label sequence is positioned5′ of the unique identifier sequence without any intervening sequencesbetween the unique molecular label sequence and the unique identifiersequence. In some embodiments, the unique molecular label sequence ispositioned 5′ of a spacer, which is positioned 5′ of the uniqueidentifier sequence, so that a spacer is between the unique molecularlabel sequence and the unique identifier sequence. In some embodiments,the unique identifier sequence is positioned 5′ of the unique molecularlabel sequence without any intervening sequences between the uniqueidentifier sequence and the unique molecular label sequence. In someembodiments, the unique identifier sequence is positioned 5′ of aspacer, which is positioned 5′ of the unique molecular label sequence,so that a spacer is between the unique identifier sequence and theunique molecular label sequence.

The unique molecular label sequence can comprise a nucleic acid sequenceof at least 3 nucleotides, for example at least 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50 nucleotides, including ranges between any two of thelisted values, for example 3-50, 3-45, 3-40, 3-35, 3-30, 3-25, 3-20,3-15, 3-14, 3-13, 3-12, 3-11, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-50,4-45, 4-40, 4-35, 4-30, 4-25, 4-20, 4-15, 4-14, 4-13, 4-12, 4-11, 4-10,4-9, 4-8, 4-7, 4-6, 4-5, 5-50, 5-45, 5-40, 5-35, 5-30, 5-25, 5-20, 5-15,5-14, 5-13, 5-12, 5-11, 5-10, 5-9, 5-8, 5-7, 5-6, 6-50, 6-45, 6-40,6-35, 6-30, 6-25, 6-20, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8,6-7, 7-50, 7-45, 7-40, 7-35, 7-30, 7-25, 7-20, 7-15, 7-14, 7-13, 7-12,7-11, 7-10, 7-9, 7-8, 8-50, 8-45, 8-40, 8-35, 8-30, 8-25, 8-20, 8-15,8-14, 8-13, 8-12, 8-11, 8-10, 8-9, 9-50, 9-45, 9-40, 9-35, 9-30, 9-25,9-20, 9-15, 9-14, 9-13, 9-12, 9-11, 9-10, 10-50, 10-45, 10-40, 10-35,10-30, 10-25, 10-20, 10-15, 10-14, 10-13, 10-12, or 10-11 nucleotides.In some embodiments, the unique molecular label sequence is 2-20nucleotides in length.

In some embodiments, the unique molecular label sequence of the bindingreagent oligonucleotide comprises the sequence of at least three repeatsof the doublets “VN” and/or “NV” (in which each “V” is any of A, C, orG, and in which “N” is any of A, G, C, or T), for example at least 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 repeats,including ranges between any two of the listed values. Examples ofmultiple repeats of the doublet “VN” include VN, VNVN, VNVNVN, andVNVNVNVN. It is noted that while the formulas “VN” and “NV” describeconstraints on the base content, not every V or every N has to be thesame or different. For example, if the molecule barcodes of uniqueoligonucleotide species in a composition comprised VNVNVN, one moleculebarcode can comprise the sequence ACGGCA, while another molecule barcodecan comprise the sequence ATACAT, while another molecule barcode couldcomprise the sequence ATACAC. It is noted that any number of repeats ofthe doublet “VN” would have a T content of no more than 50%. In someembodiments, at least 95% of the unique oligonucleotide species of acomposition comprising at least 1000 unique oligonucleotide speciescomprise molecule barcodes comprising at least three repeats of thedoublets “VN” and/or “NV,” for example at least 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 repeats, including rangesbetween any two of the listed values. In some embodiments, at least 99%of the unique oligonucleotide species of a composition comprising atleast 1000 unique oligonucleotide species comprise molecule barcodescomprising at least three repeats of the doublets “VN” and/or “NV,” forexample at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, or 20 repeats, including ranges between any two of the listedvalues. In some embodiments, at least 99.9% of the uniqueoligonucleotide species of a composition comprising at least 1000 uniqueoligonucleotide species comprise molecule barcodes comprising at leastthree repeats of the doublets “VN” and/or “NV,” for example at least 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 repeats,including ranges between any two of the listed values. In someembodiments, at least 95% of the unique oligonucleotide species of acomposition comprising at least 6500 unique oligonucleotide speciescomprise molecule barcodes comprising at least three repeats of thedoublets “VN” and/or “NV,” for example at least 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 repeats, including rangesbetween any two of the listed values. In some embodiments, at least 99%of the unique oligonucleotide species of a composition comprising atleast 6500 unique oligonucleotide species comprise molecule barcodescomprising at least three repeats of the doublets “VN” and/or “NV,” forexample at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, or 20 repeats, including ranges between any two of the listedvalues. In some embodiments, at least 99.9% of the uniqueoligonucleotide species of a composition comprising at least 6500 uniqueoligonucleotide species comprise molecule barcodes comprising at leastthree repeats of the doublets “VN” and/or “NV,” for example at least 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 repeats,including ranges between any two of the listed values. In someembodiments, at least 95% of the unique oligonucleotide species of acomposition comprising at least 65,000 unique oligonucleotide speciescomprise molecule barcodes comprising at least three repeats of thedoublets “VN” and/or “NV,” for example at least 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 repeats, including rangesbetween any two of the listed values. In some embodiments, at least 99%of the unique oligonucleotide species of a of composition comprising atleast 65,000 unique oligonucleotide species comprise molecule barcodescomprising at least three repeats of the doublets “VN” and/or “NV,” forexample at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, or 20 repeats, including ranges between any two of the listedvalues. In some embodiments, at least 99.9% of the uniqueoligonucleotide species of a composition comprising at least 65,000unique oligonucleotide species comprise molecule barcodes comprising atleast three repeats of the doublets “VN” and/or “NV,” for example atleast 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20repeats, including ranges between any two of the listed values. In someembodiments, the composition consists of or consists essentially of atleast 1000, 6500, or 65,000 unique oligonucleotide species that eachhave a molecule barcode comprising the sequence VNVNVN. In someembodiments, the composition consists of or consists essentially of atleast 1000, 6500, or 65,000 unique oligonucleotide species that each hasa molecule barcode comprising the sequence VNVNVNVN. In someembodiments, at least 95%, 99%, or 99.9% of the barcode regions of thecomposition as described herein comprise at least three repeats of thedoublets “VN” and/or “NV,” as described herein. In some embodiments,unique molecular label sequences comprising repeated “doublets “VN”and/or “NV” can yield low bias, while providing a compromise betweenreducing bias and maintaining a relatively large quantity of availablenucleotide sequences, so that relatively high diversity can be obtainedin a relatively short sequence, while still minimizing bias. In someembodiments, unique molecular label sequences comprising repeated“doublets “VN” and/or “NV” can reduce bias by increasing sensitivity,decreasing relative standard error, or increasing sensitivity andreducing standard error. In some embodiments, unique molecular labelsequences comprising repeated “doublets “VN” and/or “NV” improveinformatics analysis by serving as a geomarker. In some embodiments, therepeated doublets “VN” and/or “NV” described herein reduce the incidenceof homopolymers within the unique molecular label sequences. In someembodiments, the repeated doublets “VN” and/or “NV” described hereinbreak up homopolymers.

In some embodiments, the sample indexing oligonucleotide comprises afirst molecular label sequence. In some embodiments, the first molecularlabel sequences of at least two sample indexing oligonucleotides aredifferent, and the sample indexing sequences of the at least two sampleindexing oligonucleotides are identical. In some embodiments, the firstmolecular label sequences of at least two sample indexingoligonucleotides are different, and the sample indexing sequences of theat least two sample indexing oligonucleotides are different. In someembodiments, the cellular component-binding reagent specificoligonucleotide comprises a second molecular label sequence. In someembodiments, the second molecular label sequences of at least twocellular component-binding reagent specific oligonucleotides aredifferent, and the unique identifier sequences of the at least twocellular component-binding reagent specific oligonucleotides areidentical. In some embodiments, the second molecular label sequences ofat least two cellular component-binding reagent specificoligonucleotides are different, and the unique identifier sequences ofthe at least two cellular component-binding reagent specificoligonucleotides are different. In some embodiments, the number ofunique second molecular label sequences associated with the uniqueidentifier sequence for the cellular component-binding reagent capableof specifically binding to the at least one cellular component target inthe sequencing data indicates the number of copies of the at least onecellular component target in the one or more of the plurality of cells.In some embodiment, a combination (e.g., minimum, average, and maximum)of (1) the number of unique first molecular label sequences associatedwith the unique identifier sequence for the cellular component-bindingreagent capable of specifically binding to the at least one cellularcomponent target in the sequencing data and (2) the number of uniquesecond molecular label sequences associated with the unique identifiersequence for the cellular component-binding reagent capable ofspecifically binding to the at least one cellular component target inthe sequencing data indicates the number of copies of the at least onecellular component target in the one or more of the plurality of cells.

Alignment Sequence

In some embodiments, the binding reagent oligonucleotide comprises analignment sequence (e.g., the alignment sequence 825 bb described withreference to FIG. 9) adjacent to the poly(dA) region. The alignmentsequence can be 1 or more nucleotides in length. The alignment sequencecan be 2 nucleotides in length. The alignment sequence can comprise aguanine, a cytosine, a thymine, a uracil, or a combination thereof. Thealignment sequence can comprise a poly(dT) region, a poly(dG) region, apoly(dC) region, a poly(dU) region, or a combination thereof. In someembodiments, the alignment sequence is 5′ to the poly(dA) region.Advantageously, in some embodiments, the presence of the alignmentsequence enables the poly(A) tail of each of the binding reagentoligonucleotides to have the same length, leading to greater uniformityof performance. In some embodiments, the percentage of binding reagentoligonucleotides with an identical poly(dA) region length within aplurality of binding reagent oligonucleotides, each of which comprise analignment sequence, can be, or be about, 80%, 90%, 91%, 93%, 95%, 97%,99.9%, 99.9%, 99.99%, or 100%, or a number or a range between any two ofthese values. In some embodiments, the percentage of binding reagentoligonucleotides with an identical poly(dA) region length within theplurality of binding reagent oligonucleotides, each of which comprise analignment sequence, can be at least, or be at most, 80%, 90%, 91%, 93%,95%, 97%, 99.9%, 99.9%, 99.99%, or 100%.

The length of the alignment sequence can be different in differentimplementations. In some embodiments, the length of the alignmentsequence can be, or can be about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or anumber or a range between any two of these values. In some embodiments,the length of the alignment sequence can be at least, or can be at most,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, or 100. The number of guanine(s),cytosine(s), thymine(s), or uracil(s) in the alignment sequence can bedifferent in different implementations. The number of guanine(s),cytosine(s), thymine(s), or uracil(s) can be, or can be about, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, or a number or a range between any two of thesevalues. The number of guanine(s), cytosine(s), thymine(s), or uracil(s)can be at least, or can be at most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.In some embodiments, the sample indexing oligonucleotide comprises analignment sequence. In some embodiments, the cellular component-bindingreagent specific oligonucleotide comprises an alignment sequence.

Linker

The binding reagent oligonucleotide can be conjugated with the cellularcomponent binding reagent through various mechanisms. In someembodiments, the binding reagent oligonucleotide can be conjugated withthe cellular component binding reagent covalently. In some embodiments,the binding reagent oligonucleotide can be conjugated with the cellularcomponent binding reagent non-covalently. In some embodiments, thebinding reagent oligonucleotide is conjugated with the cellularcomponent binding reagent through a linker. In some embodiments, thebinding reagent oligonucleotide can comprise the linker. The linker cancomprise a chemical group. The chemical group can be reversibly, orirreversibly, attached to the molecule of the cellular component bindingreagent. The chemical group can be selected from the group consisting ofa UV photocleavable group, a disulfide bond, a streptavidin, a biotin,an amine, and any combination thereof. The linker can comprise a carbonchain. The carbon chain can comprise, for example, 5-50 carbon atoms.The carbon chain can have different numbers of carbon atoms in differentembodiments. In some embodiments, the number of carbon atoms in thecarbon chain can be, or can be about, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, or a number or a range between any two of these values. In someembodiments, the number of carbon atoms in the carbon chain can be atleast, or can be at most, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.In some embodiments, the carbon chain comprises 2-30 carbons. In someembodiments, the carbon chain comprises 12 carbons. In some embodiments,amino modifiers employed for binding reagent oligonucleotide can beconjugated to the cellular component binding reagent. In someembodiments, the linker comprises 5′ amino modifier C6 (5AmMC6). In someembodiments, the linker comprises 5′ amino modifier C12 (5AmMC12). Insome embodiments, the linker comprises a derivative of 5AmMC12. In someembodiments, a longer linker achieves a higher efficiency ofconjugation. In some embodiments, a longer linker achieves a higherefficiency of modification prior to conjugation. In some embodiments,increasing the distance between the functional amine and the DNAsequence yields a higher efficiency of conjugation. In some embodiments,increasing the distance between the functional amine and the DNAsequence yields a higher efficiency of modification prior toconjugation. In some embodiments, the use of 5AmMC12 as a linker yieldsa higher efficiency of modification (prior to conjugation) than the useof 5AmMC6 as a linker. In some embodiments the use of 5AmMC12 as alinker yields a higher efficiency of conjugation than the use of 5AmMC6as a linker. In some embodiments, the sample indexing oligonucleotide isassociated with the cellular component-binding reagent through a linker.In some embodiments, the cellular component-binding reagent specificoligonucleotide is associated with the cellular component-bindingreagent through a linker.

Antibody-Specific Barcode Sequence

Disclosed herein, in several embodiments, are improvements to the designof the unique identifier sequence (e.g., antibody-specific barcodesequence) of a binding reagent oligonucleotide. In some embodiments theunique identifier sequence (e.g, sample indexing sequence, cellularcomponent-binding reagent specific oligonucleotide) is designed to havea Hamming distance greater than 3. In some embodiments, the Hammingdistance of the unique identifier sequence can be, or be about, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, or a number or a range between any two of thesevalues. In some embodiments, the unique identifier sequences has a GCcontent in the range of 40% to 60% and does not have a predictedsecondary structure (e.g., hairpin). In some embodiments, the uniqueidentifier sequence does not comprise any sequences predicted in silicoto bind to the mouse and/or human transcripts. In some embodiments, theunique identifier sequence does not comprise any sequences predicted insilico to bind to Rhapsody and/or SCMK system primers. In someembodiments, the unique identifier sequence does not comprisehomopolymers.

Primer Adapter

In some embodiments, the binding reagent oligonucleotide comprises aprimer adapter. In some embodiments, the primer adapter comprises thesequence of a first universal primer, a complimentary sequence thereof,a partial sequence thereof, or a combination thereof. In someembodiments, the first universal primer comprises an amplificationprimer, a complimentary sequence thereof, a partial sequence thereof, ora combination thereof. In some embodiments, the first universal primercomprises a sequencing primer, a complimentary sequence thereof, apartial sequence thereof, or a combination thereof. In some embodiments,the sequencing primer comprises an Illumina sequencing primer. In someembodiments, the sequencing primer comprises a portion of an Illuminasequencing primer. In some embodiments, the sequencing primer comprisesa P7 sequencing primer. In some embodiments, the sequencing primercomprises a portion of P7 sequencing primer. In some embodiments, theprimer adapter comprises an adapter for Illumina P7. In someembodiments, the primer adapter comprises a partial adapter for IlluminaP7. In some embodiments, the amplification primer is an Illumina P7sequence or a subsequence thereof. In some embodiments, the sequencingprimer is an Illumina R2 sequence or a subsequence thereof. In someembodiments, the first universal primer is 5-50 nucleotides in length.In some embodiments. The primer adapter can comprise a nucleic acidsequence of at least 5 nucleotides, for example at least 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, or 50 nucleotides, including ranges between any two ofthe listed values, for example 5-50, 5-45, 5-40, 5-35, 5-30, 5-25, 5-20,5-15, 5-14, 5-13, 5-12, 5-11, 5-10, 5-9, 5-8, 5-7, 5-6, 6-50, 6-45,6-40, 6-35, 6-30, 6-25, 6-20, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9,6-8, 6-7, 7-50, 7-45, 7-40, 7-35, 7-30, 7-25, 7-20, 7-15, 7-14, 7-13,7-12, 7-11, 7-10, 7-9, 7-8, 8-50, 8-45, 8-40, 8-35, 8-30, 8-25, 8-20,8-15, 8-14, 8-13, 8-12, 8-11, 8-10, 8-9, 9-50, 9-45, 9-40, 9-35, 9-30,9-25, 9-20, 9-15, 9-14, 9-13, 9-12, 9-11, 9-10, 10-50, 10-45, 10-40,10-35, 10-30, 10-25, 10-20, 10-15, 10-14, 10-13, 10-12, or 10-11nucleotides. The primer adapter can comprise a nucleic acid sequence ofat least 5 nucleotides of the sequence of a first universal primer, anamplification primer, a sequencing primer, a complimentary sequencethereof, a partial sequence thereof, or a combination thereof, forexample at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides,including ranges between any two of the listed values, for example 5-50,5-45, 5-40, 5-35, 5-30, 5-25, 5-20, 5-15, 5-14, 5-13, 5-12, 5-11, 5-10,5-9, 5-8, 5-7, 5-6, 6-50, 6-45, 6-40, 6-35, 6-30, 6-25, 6-20, 6-15,6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 6-7, 7-50, 7-45, 7-40, 7-35,7-30, 7-25, 7-20, 7-15, 7-14, 7-13, 7-12, 7-11, 7-10, 7-9, 7-8, 8-50,8-45, 8-40, 8-35, 8-30, 8-25, 8-20, 8-15, 8-14, 8-13, 8-12, 8-11, 8-10,8-9, 9-50, 9-45, 9-40, 9-35, 9-30, 9-25, 9-20, 9-15, 9-14, 9-13, 9-12,9-11, 9-10, 10-50, 10-45, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15,10-14, 10-13, 10-12, or 10-11 nucleotides of the sequence of a firstuniversal primer, an amplification primer, a sequencing primer, acomplimentary sequence thereof, a partial sequence thereof, or acombination thereof.

In some embodiments, the oligonucleotide barcodes provided hereincomprise a first universal sequence and the binding reagentoligonucleotides comprise a second universal sequence (e.g., a primeradapter). The first universal sequence and the second universal sequencecan be the same or different. The first universal sequence and/or thesecond universal sequence can comprise the binding sites of a sequencingprimer, a sequencing adaptor, complementary sequences thereof, and/orportions thereof. The sequencing adaptor can comprise a P5 sequence, aP7 sequence, complementary sequences thereof, and/or portions thereof.The sequencing primer can comprise a Read 1 sequencing primer, a Read 2sequencing primer, complementary sequences thereof, and/or portionsthereof.

A conventional amplification workflow for sequencing library preparationcan employ three rounds of PCR, such as, for example: a first round(“PCR 1”) employing a target-specific primer and a primer against theuniversal Illumina sequencing primer 1 sequence; a second round (“PCR2”) using a nested target-specific primer flanked by Illumina sequencingprimer 2 sequence, and a primer against the universal Illuminasequencing primer 1 sequence; and a third round (“PCR 3”) addingIllumina P5 and P7 and sample index. Advantageously, in someembodiments, the primer adapter disclosed herein enables a shorter andsimpler workflow in library preparation as compared to if the startingtemplate (e.g., a sample indexing oligonucleotide attached to a bead)does not have a primer adapter. In some embodiments, the primer adapterreduces pre-sequencing PCR amplification of a template by one round (ascompared to if the template does not comprise a primer adapter). In someembodiments, the primer adapter reduces pre-sequencing PCR amplificationof the template to one round (as compared to if the template does notcomprise a primer adapter). In some embodiments, a template comprisingthe primer adapter does not require a PCR amplification step forattachment of Illumina sequencing adapters that would be requiredpre-sequencing if the template did not comprise a primer adapter. Insome embodiments, the primer adapter sequence (or a subsequence thereof)is not part of the sequencing readout of a sequencing templatecomprising a primer adapter sequence and therefore does not affect readquality of a template comprising a primer adapter. In some embodiments,a template comprising the primer adapter has decreased sequencingdiversity as compared to if the template does not comprise a primeradapter.

In some embodiments, the sample indexing oligonucleotide comprises aprimer adapter. In some embodiments, replicating a sample indexingoligonucleotide, a barcoded sample indexing oligonucleotide, or aproduct thereof, comprises using a first universal primer, a firstprimer comprising the sequence of the first universal primer, or acombination thereof, to generate a plurality of replicated sampleindexing oligonucleotides. In some embodiments, replicating a one sampleindexing oligonucleotide, a barcoded sample indexing oligonucleotide, ora product thereof, comprises using a first universal primer, a firstprimer comprising the sequence of the first universal primer, a seconduniversal primer, a second primer comprising the sequence of the seconduniversal primer, or a combination thereof, to generate the plurality ofreplicated sample indexing oligonucleotides. In some embodiments, thecellular component-binding reagent specific oligonucleotide comprises aprimer adapter. In some embodiments, the cellular component-bindingreagent specific oligonucleotide comprises the sequence of a firstuniversal primer, a complementary sequence thereof, a partial sequencethereof, or a combination thereof

Binding Reagent Oligonucleotide Barcoding

FIG. 8 shows a schematic illustration of a non-limiting exemplaryworkflow of barcoding of a binding reagent oligonucleotide 825 (antibodyoligonucleotide illustrated here) that is associated with a bindingreagent 805 (antibody illustrated here). The binding reagentoligonucleotide 825 can be associated with binding reagent 805 throughlinker 8251. The binding reagent oligonucleotide 825 can be detachedfrom the binding reagent using chemical, optical or other means. Thebinding reagent oligonucleotide 825 can be an mRNA mimic. The bindingreagent oligonucleotide 825 can include a primer adapter 825 pa, anantibody molecular label 825 am (e.g., a unique molecular labelsequence), an antibody barcode 825 ab (e.g., a unique identifiersequence), an alignment sequence 825 bb, and a poly(A) tail 825 a. Insome embodiments, the primer adapter 825 pa comprises the sequence of afirst universal primer, a complimentary sequence thereof, a partialsequence thereof, or a combination thereof. In some embodiments, theprimer adapter 825 pa can be the same for all or some of binding reagentoligonucleotides 825. In some embodiments, the antibody barcode 825 abcan be the same for all or some of binding reagent oligonucleotides 825.In some embodiments, the antibody barcode 825 ab of different bindingreagent oligonucleotides 825 are different. In some embodiments, theantibody molecular label 825 am of different binding reagentoligonucleotides 825 are different.

The binding reagent oligonucleotides 825 can be barcoded using aplurality of barcodes 815 (e.g., barcodes 815 associated with aparticle, such as a bead 810) to create a plurality of barcoded bindingreagent oligonucleotides 840. In some embodiments, a barcode 815 caninclude a poly(dT) region 815 t for binding to a binding reagentoligonucleotide 825, optionally a molecular label 815 m (e.g., fordetermining the number of occurrences of the binding reagentoligonucleotides), a cell label 815 c, and a universal label 815 u. Insome embodiments the barcode 815 is hybridized to the poly(dT) region815 t of binding reagent oligonucleotides 825. In some embodimentsbarcoded binding reagent oligonucleotides 840 are generated by extending(e.g., by reverse transcription) the barcode 815 hybridized to thebinding reagent oligonucleotide 825. In some embodiments, barcodedbinding reagent oligonucleotides 840 comprise primer adapter 825 pa, anantibody molecular label 825 am (e.g., a unique molecular labelsequence), an antibody barcode 825 ab (e.g., a unique identifiersequence), an alignment sequence 825 bb, poly(dT) region 815 t,molecular label 815 m, cell label 815 c, and universal label 815 u.

In some embodiments, the barcoded binding reagent oligonucleotidesdisclosed herein comprises two unique molecular label sequences: amolecular label sequence derived from the barcode (e.g., molecular label815 m) and a molecular label sequence derived from a binding reagentoligonucleotide (e.g., antibody molecular label 825 am, the firstmolecular label sequence of a sample indexing oligonucleotide, thesecond molecular label sequence of a cellular component-binding reagentspecific oligonucleotide). As used herein, “dual molecular indexing”refers to methods and compositions disclosed herein employing barcodedbinding reagent oligonucleotides (or products thereof) that comprise afirst unique molecular label sequence and second unique molecular labelsequence (or complementary sequences thereof). In some embodiments, themethods of sample identification and of quantitative analysis ofcellular component targets disclosed herein can comprise obtaining thesequence of information of the barcode molecular label sequence and/orthe binding reagent oligonucleotide molecular label sequence. In someembodiments, the number of barcode molecular label sequences associatedwith the unique identifier sequence for the cellular component-bindingreagent capable of specifically binding to the at least one cellularcomponent target in the sequencing data indicates the number of copiesof the at least one cellular component target in the one or more of theplurality of cells. In some embodiments, the number of binding reagentoligonucleotide molecular label sequences associated with the uniqueidentifier sequence for the cellular component-binding reagent capableof specifically binding to the at least one cellular component target inthe sequencing data indicates the number of copies of the at least onecellular component target in the one or more of the plurality of cells.In some embodiments, the number of both the binding reagentoligonucleotide molecular label sequences and barcode molecular labelsequences associated with the unique identifier sequence for thecellular component-binding reagent capable of specifically binding tothe at least one cellular component target in the sequencing dataindicates the number of copies of the at least one cellular componenttarget in the one or more of the plurality of cells

The use of PCR to amplify the amount of material before starting thesequencing protocol adds the potential for artifacts, such asartifactual recombination during amplification occurs when prematuretermination products prime a subsequent round of synthesis). In someembodiments, the methods of dual molecular indexing provided hereinallow the identification of PCR chimeras given sufficient sequencingdepth. Additionally, in some embodiments, the addition of the uniquemolecular label sequence to the binding reagent oligonucleotideincreases stochastic labelling complexity. Thus, in some embodiments,the presence of the unique molecular label sequence in the bindingreagent oligonucleotide can overcome UMI diversity limitations. In someembodiments the methods of dual molecular indexing provided hereindecrease the number of cellular component targets flagged as “Saturated”during post-sequencing molecular coverage calculations by at least about2% (e.g., 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,16%, 17%, 18%, 19%, 20%, 25%, 30%, 40%, 50%, 75%, 100%, 150%, 200%,250%, 500%, 1000%, or higher and overlapping ranges therein) compared toif the methods and compositions are not used.

EXAMPLES

Some aspects of the embodiments discussed above are disclosed in furtherdetail in the following examples, which are not in any way intended tolimit the scope of the present disclosure.

Example 1 Oligonucleotides for Associating with Protein Binding Reagents

This example demonstrates designing of oligonucleotides that can beconjugated with protein binding reagents. The oligonucleotides can beused to determine protein expression and gene expression simultaneously.The oligonucleotides can also be used for sample indexing to determinecells of the same or different samples.

95mer Oligonucleotide Design

The following method was used to generate candidate oligonucleotidesequences and corresponding primer sequences for simultaneousdetermination of protein expression and gene expression or sampleindexing.

1. Sequence Generation and Elimination

The following process was used to generate candidate oligonucleotidesequences for simultaneous determination of protein expression and geneexpression or sample indexing.

Step 1a. Randomly generate a number of candidate sequences (50000sequences) with the desired length (45 bps).

Step 1b. Append the transcriptional regulator LSRR sequence to the 5′end of the sequences generated and a poly(A) sequence (25 bps) to the 3′end of the sequences generated.

Step 1c. Remove sequences generated and appended that do not have GCcontents in the range of 40% to 50%.

Step 1d. Remove remaining sequences with one or more hairpin structureseach.

The number of remaining candidate oligonucleotide sequences was 423.

2. Primer Design

The following method was used to design primers for the remaining 423candidate oligonucleotide sequences.

2.1 N1 Primer:

Use the universal N1 sequence: 5′-GTTGTCAAGATGCTACCGTTCAGAG-3′ (LSRRsequence; SEQ ID NO. 3) as the N1 primer.

2.2 N2 Primer

(for amplifying specific sample index oligonucleotides; e.g., N2 primerin FIGS. 9B-9D):

2.2a. Remove candidate N2 primers that do not start downstream of the N1sequence.

2.2b. Remove candidate N2 primers that overlap in the last 35 bps of thecandidate oligonucleotide sequence.

2.2c. Remove the primer candidates that are aligned to the transcriptomeof the species of cells being studied using the oligonucleotides (e.g.,the human transcriptome or the mouse transcriptome).

2.2d. Use the ILR2 sequence as the default control(ACACGACGCTCTTCCGATCT; SEQ ID NO. 4) to minimize or avoid primer-primerinteractions.

Of the 423 candidate oligonucleotide sequences, N2 primers for 390candidates were designed.

3. Filtering

The following process was used to filter the remaining 390 candidateprimer sequences.

3a. Eliminate any candidate oligonucleotide sequence with a randomsequence ending in As (i.e., the effective length of the poly(A)sequence is greater than 25 bps) to keep poly(A) tail the same lengthfor all barcodes.

3b. Eliminate any candidate oligonucleotide sequences with 4 or moreconsecutive Gs (>3Gs) because of extra cost and potentially lower yieldin oligo synthesis of runs of Gs.

FIG. 9A shows a non-limiting exemplary candidate oligonucleotidesequence generated using the method above.

200mer Oligonucleotide Design

The following method was used to generate candidate oligonucleotidesequences and corresponding primer sequences for simultaneousdetermination of protein expression and gene expression and sampleindexing.

1. Sequence Generation and Elimination

The following was used to generate candidate oligonucleotide sequencesfor simultaneous determination of protein expression and gene expressionand sample indexing.

1a. Randomly generate a number of candidate sequences (100000 sequences)with the desired length (128 bps).

1b. Append the transcriptional regulator LSRR sequence and an additionalanchor sequence that is non-human, non-mouse to the 5′ end of thesequences generated and a poly(A) sequence (25 bps) to the 3′ end of thesequences generated.

1c. Remove sequences generated and appended that do not have GC contentsin the range of 40% to 50%.

1d. Sort remaining candidate oligonucleotide sequences based on hairpinstructure scores.

1e. Select 1000 remaining candidate oligonucleotide sequences with thelowest hairpin scores.

2. Primer Design

The following method was used to design primers for 400 candidateoligonucleotide sequences with the lowest hairpin scores.

2.1 N1 Primer:

Use the universal N1 sequence: 5′-GTTGTCAAGATGCTACCGTTCAGAG-3′ (LSRRsequence; SEQ ID NO. 3) as the N1 primer.

2.2 N2 Primer

(for amplifying specific sample index oligonucleotides; e.g., N2 primerin FIGS. 9B and 9C):

2.2a. Remove candidate N2 primers that do not start 23 nts downstream ofthe N1 sequence (The anchor sequence was universal across all candidateoligonucleotide sequences).

2.2b. Remove candidate N2 primers that overlap in the last 100 bps ofthe target sequence. The resulting primer candidates can be between the48th nucleotide and 100th nucleotide of the target sequence.

2.2c. Remove the primer candidates that are aligned to the transcriptomeof the species of cells being studied using the oligonucleotides (e.g.,the human transcriptome or the mouse transcriptome).

2.2d. Use the ILR2 sequence, 5′-ACACGACGCTCTTCCGATCT-3′ (SEQ ID NO. 4)as the default control to minimize or avoid primer-primer interactions.

2.2e. Remove N2 primer candidates that overlap in the last 100 bps ofthe target sequence.

Of the 400 candidate oligonucleotide sequences, N2 primers for 392candidates were designed.

3. Filtering

The following was used to filter the remaining 392 candidate primersequences.

3a. Eliminate any candidate oligonucleotide sequence with a randomsequence ending in As (i.e., the effective length of the poly(A)sequence is greater than 25 bps) to keep poly(A) tail the same lengthfor all barcodes.

3b. Eliminate any candidate oligonucleotide sequences with 4 or moreconsecutive Gs (>3 Gs) because of extra cost and potentially lower yieldin oligo synthesis of runs of Gs.

FIG. 9B shows a non-limiting exemplary candidate oligonucleotidesequence generated using the method above. The nested N2 primer shown inFIG. 9B can bind to the antibody or sample specific sequence fortargeted amplification. FIG. 9C shows the same non-limiting exemplarycandidate oligonucleotide sequence with a nested universal N2 primerthat corresponds to the anchor sequence for targeted amplification. FIG.9D shows the same non-limiting exemplary candidate oligonucleotidesequence with a N2 primer for one step targeted amplification.

Altogether, these data indicate that oligonucleotide sequences ofdifferent lengths can be designed for simultaneous determination ofprotein expression and gene expression or sample indexing. Theoligonucleotide sequences can include a universal primer sequence, anantibody specific oligonucleotide sequence or a sample indexingsequence, and a poly(A) sequence.

Example 2 Oligonucleotide-Associated Antibody Workflow

This example demonstrates a workflow of using anoligonucleotide-conjugated antibody for determining the expressionprofile of a protein target.

Frozen cells (e.g., frozen peripheral blood mononuclear cells (PBMCs))of a subject are thawed. The thawed cells are stained with anoligonucleotide-conjugated antibody (e.g., an anti-CD4 antibody at 0.06μg/100 μl (1:333 dilution of an oligonucleotide-conjugated antibodystock)) at a temperature for a duration (e.g., room temperature for 20minutes). The oligonucleotide-conjugated antibody is conjugated with 1,2, or 3 oligonucleotides (“antibody oligonucleotides”). The sequence ofthe antibody oligonucleotide is shown in FIG. 10. The cells are washedto remove unbound oligonucleotide-conjugated antibody. The cells areoptionally stained with Calcein AM (BD (Franklin Lake, N.J.)) and Drag7™(Abcam (Cambridge, United Kingdom)) for sorting with flow cytometry toobtain cells of interest (e.g., live cells). The cells are optionallywashed to remove excess Calcein AM and Drag7™. Single cells stained withCalcein AM (live cells) and not Drag7™ (cells that are not dead orpermeabilized) are sorted, using flow cytometry, into a BD Rhapsody™cartridge.

Of the wells containing a single cell and a bead, the single cells inthe wells (e.g., 3500 live cells) are lysed in a lysis buffer (e.g., alysis buffer with 5 mM DTT). The mRNA expression profile of a target(e.g., CD4) is determined using BD Rhapsody™ beads. The proteinexpression profile of a target (e.g., CD4) is determined using BDRhapsody™ beads and the antibody oligonucleotides. Briefly, the mRNAmolecules are released after cell lysis. The Rhapsody™ beads areassociated with barcodes (e.g., stochastic barcodes) each containing amolecular label, a cell label, and an oligo(dT) region. The poly(A)regions of the mRNA molecules released from the lysed cells hybridize tothe poly(T) regions of the stochastic barcodes. The poly(dA) regions ofthe antibody oligonucleotides hybridize to the oligo(dT) regions of thebarcodes. The mRNA molecules were reverse transcribed using thebarcodes. The antibody oligonucleotides are replicated using thebarcodes. The reverse transcription and replication optionally occur inone sample aliquot at the same time.

The reverse transcribed products and replicated products are PCRamplified using primers for determining mRNA expression profiles ofgenes of interest, using N1 primers, and the protein expression profileof a target, using the antibody oligonucleotide N1 primer. For example,the reverse transcribe products and replicated products can be PCRamplified for 15 cycles at 60 degrees annealing temperature usingprimers for determining the mRNA expression profiles of 488 blood panelgenes, using blood panel N1 primers, and the expression profile of CD4protein, using the antibody oligonucleotide N1 primer (“PCR F”). Excessbarcodes are optionally removed with Ampure cleanup. The products fromPCR 1 are optionally divided into two aliquots, one aliquot fordetermining the mRNA expression profiles of the genes of interest, usingthe N2 primers for the genes of interest, and one aliquot fordetermining the protein expression profile of the target of interest,using the antibody oligonucleotide N2 primer (“PCR 2”). Both aliquotsare PCR amplified (e.g., for 15 cycles at 60 degrees annealingtemperature). The protein expression of the target in the cells aredetermined based on the antibody oligonucleotides as illustrated in FIG.10 (“PCR 2”). Sequencing data is obtained and analyzed after sequencingadaptor addition (“PCR 3”), such as sequencing adaptor ligation. Celltypes are determined based on the mRNA expression profiles of the genesof interest.

Altogether, this example describes using an oligonucleotide-Conjugatedantibody for determining the protein expression profile of a target ofinterest. This example further describes that the protein expressionprofile of the target of interest and the mRNA expression profiles ofgenes of interest can be determine simultaneously.

Example 3 Cellular Component-Binding Reagent Oligonucleotides

FIGS. 11A-11B show non-limiting exemplary designs of oligonucleotidesfor determining protein expression and gene expression simultaneouslyand for sample indexing. FIG. 11A shows a non-limiting exemplarycellular component-binding reagent oligonucleotide (SEQ ID NO: 7)comprising a 5′ amino modifier C6 (5AmMC6) linker for antibodyconjugation (e.g., can be modified prior to antibody conjugation), auniversal PCR handle, an antibody-specific barcode sequence, and apoly(A) tail. While this embodiment depicts a poly(A) tail that is 25nucleotides long, the length of the poly(A) tail can vary. In someembodiments, the antibody-specific barcode sequence is antibodyclone-specific barcode for use in methods of protein expressionprofiling. In some embodiments, the antibody-specific barcode sequenceis a sample tag sequence for use in methods of sample indexing.Exemplary design characteristics of the antibody-specific barcodesequence are, in some embodiments, a Hamming distance greater than 3, aGC content in the range of 40% to 60%, and an absence of predictedsecondary structures (e.g., hairpin). In some embodiments, the universalPCR handle is employed for targeted PCR amplification during librarypreparation that attaches Illumina sequencing adapters to the amplicons.In some embodiments, high quality sequencing reads can be achieved byreducing sequencing diversity.

FIG. 11B shows a non-limiting exemplary cellular component-bindingreagent oligonucleotide (SEQ ID NO: 8) comprising a 5′ amino modifierC12 (5AmMC12) linker for antibody conjugation, a primer adapter (e.g., apartial adapter for Illumina P7), an antibody unique molecularidentifier (UMI), an antibody-specific barcode sequence, an alignmentsequence, and a poly(A) tail. While this embodiment depicts a poly(A)tail that is 25 nucleotides long, the length of the poly(A) tail canrange, in some embodiments, from 18-30 nucleotides. Exemplary designcharacteristics of the antibody-specific barcode sequence (wherein “X”indicates any nucleotide), in addition to those described in FIG. 11A,include, in some embodiments, an absence of homopolymers and an absenceof sequences predicted in silico to bind human transcripts, mousetranscripts, Rhapsody system primers, and/or SCMK system primers. Insome embodiments, the alignment sequence comprises the sequence BB (inwhich B is C, G, or T). Alignment sequences 1 nucleotide in length andmore than 2 nucleotides in length are provided in some embodiments. The5AmMC12 linker, can, in some embodiments, achieve a higher efficiency(e.g., for antibody conjugation or the modification prior to antibodyconjugation) as compared to a shorter linker (e.g., 5AmMC6). Theantibody UMI sequence can comprise “VN” and/or “NV” doublets (in whicheach “V” is any of A, C, or G, and in which “N” is any of A, G, C, orT), which, in some embodiments, improve informatics analysis by servingas a geomarker and/or reduce the incidence of homopolymers. In someembodiments, the presence of a unique molecular labeling sequence on thebinding reagent oligonucleotide increases stochastic labellingcomplexity. In some embodiments, the primer adapter comprises thesequence of a first universal primer, a complimentary sequence thereof,a partial sequence thereof, or a combination thereof. In someembodiments, the primer adapter eliminates the need for a PCRamplification step for attachment of Illumina sequencing adapters thatwould typically be required before sequencing. In some embodiments, theprimer adapter sequence (or a subsequence thereof) is not part of thesequencing readout of a sequencing template comprising a primer adaptersequence and therefore does not affect read quality of a templatecomprising a primer adapter.

Terminology

In at least some of the previously described embodiments, one or moreelements used in an embodiment can interchangeably be used in anotherembodiment unless such a replacement is not technically feasible. Itwill be appreciated by those skilled in the art that various otheromissions, additions and modifications may be made to the methods andstructures described above without departing from the scope of theclaimed subject matter. All such modifications and changes are intendedto fall within the scope of the subject matter, as defined by theappended claims.

With respect to the use of substantially any plural and/or singularterms herein, those having skill in the art can translate from theplural to the singular and/or from the singular to the plural as isappropriate to the context and/or application. The varioussingular/plural permutations may be expressly set forth herein for sakeof clarity. As used in this specification and the appended claims, thesingular forms “a,” “an,” and “the” include plural references unless thecontext clearly dictates otherwise. Any reference to “or” herein isintended to encompass “and/or” unless otherwise stated.

It will be understood by those within the art that, in general, termsused herein, and especially in the appended claims (e.g., bodies of theappended claims) are generally intended as “open” terms (e.g., the term“including” should be interpreted as “including but not limited to,” theterm “having” should be interpreted as “having at least,” the term“includes” should be interpreted as “includes but is not limited to,”etc.). It will be further understood by those within the art that if aspecific number of an introduced claim recitation is intended, such anintent will be explicitly recited in the claim, and in the absence ofsuch recitation no such intent is present. For example, as an aid tounderstanding, the following appended claims may contain usage of theintroductory phrases “at least one” and “one or more” to introduce claimrecitations. However, the use of such phrases should not be construed toimply that the introduction of a claim recitation by the indefinitearticles “a” or “an” limits any particular claim containing suchintroduced claim recitation to embodiments containing only one suchrecitation, even when the same claim includes the introductory phrases“one or more” or “at least one” and indefinite articles such as “a” or“an” (e.g., “a” and/or “an” should be interpreted to mean “at least one”or “one or more”); the same holds true for the use of definite articlesused to introduce claim recitations. In addition, even if a specificnumber of an introduced claim recitation is explicitly recited, thoseskilled in the art will recognize that such recitation should beinterpreted to mean at least the recited number (e.g., the barerecitation of “two recitations,” without other modifiers, means at leasttwo recitations, or two or more recitations). Furthermore, in thoseinstances where a convention analogous to “at least one of A, B, and C,etc.” is used, in general such a construction is intended in the senseone having skill in the art would understand the convention (e.g., “asystem having at least one of A, B, and C” would include but not belimited to systems that have A alone, B alone, C alone, A and Btogether, A and C together, B and C together, and/or A, B, and Ctogether, etc.). In those instances where a convention analogous to “atleast one of A, B, or C, etc.” is used, in general such a constructionis intended in the sense one having skill in the art would understandthe convention (e.g., “a system having at least one of A, B, or C” wouldinclude but not be limited to systems that have A alone, B alone, Calone, A and B together, A and C together, B and C together, and/or A,B, and C together, etc.). It will be further understood by those withinthe art that virtually any disjunctive word and/or phrase presenting twoor more alternative terms, whether in the description, claims, ordrawings, should be understood to contemplate the possibilities ofincluding one of the terms, either of the terms, or both terms. Forexample, the phrase “A or B” will be understood to include thepossibilities of “A” or “B” or “A and B.”

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

As will be understood by one skilled in the art, for any and allpurposes, such as in terms of providing a written description, allranges disclosed herein also encompass any and all possible sub-rangesand combinations of sub-ranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” and the likeinclude the number recited and refer to ranges which can be subsequentlybroken down into sub-ranges as discussed above. Finally, as will beunderstood by one skilled in the art, a range includes each individualmember. Thus, for example, a group having 1-3 articles refers to groupshaving 1, 2, or 3 articles. Similarly, a group having 1-5 articlesrefers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

While various aspects and embodiments have been disclosed herein, otheraspects and embodiments will be apparent to those skilled in the art.The various aspects and embodiments disclosed herein are for purposes ofillustration and are not intended to be limiting, with the true scopeand spirit being indicated by the following claims.

What is claimed is:
 1. A composition comprising: a plurality of cellularcomponent-binding reagents each associated with a cellularcomponent-binding reagent specific oligonucleotide comprising a uniqueidentifier sequence for the cellular component-binding reagent, whereinthe cellular component-binding reagent is capable of specificallybinding to at least one of a plurality of cellular component targets,and wherein the cellular component-binding reagent specificoligonucleotide comprises one or more of: (a) a first molecular labelsequence; (b) an alignment sequence adjacent to a poly(dA) region; (c) alinker, wherein the cellular component-binding reagent specificoligonucleotide is associated with the cellular component-bindingreagent through the linker; and (d) the sequence of a first universalprimer, a complementary sequence thereof, a partial sequence thereof, ora combination thereof.
 2. The composition of claim 1, wherein the firstmolecular label sequence is 2-20 nucleotides in length.
 3. Thecomposition of claim 1, wherein the first molecular label sequences ofat least two cellular component-binding reagent specificoligonucleotides are different, and wherein the unique identifiersequences of the at least two cellular component-binding reagentspecific oligonucleotides are identical.
 4. The composition of claim 1,wherein the first molecular label sequences of at least two cellularcomponent-binding reagent specific oligonucleotides are different, andwherein the unique identifier sequences of the at least two cellularcomponent-binding reagent specific oligonucleotides are different. 5.The composition of claim 1, wherein the first universal primer is 5-50nucleotides in length.
 6. The composition of claim 1, wherein the firstuniversal primer comprises an amplification primer, a sequencing primer,or a combination thereof.
 7. The composition of claim 6, wherein thesequencing primer comprises a P7 sequencing primer.
 8. The compositionof claim 1, wherein the alignment sequence is one or more nucleotides inlength.
 9. The composition of claim 1, wherein the alignment sequence istwo or more nucleotides in length.
 10. The composition of claim 1,wherein the alignment sequence comprises a guanine, a cytosine, athymine, a uracil, or a combination thereof.
 11. The composition ofclaim 1, wherein the alignment sequence comprises a poly(dT) sequence, apoly(dG) sequence, a poly(dC) sequence, a poly(dU) sequence, or acombination thereof.
 12. The composition of claim 1, wherein thealignment sequence is 5′ to the poly(dA) region.
 13. The composition ofclaim 1, wherein the linker comprises a carbon chain.
 14. Thecomposition of claim 13, wherein the carbon chain comprises 2-30carbons.
 15. The composition of claim 1, wherein the linker comprises 5′amino modifier C12 (5AmMC12), or a derivative thereof.
 16. A method formeasuring cellular component expression in cells, comprising: contactinga plurality of cellular component-binding reagents with a plurality ofcells comprising a plurality of cellular component targets, wherein eachof the plurality of cellular component-binding reagents comprises acellular component-binding reagent specific oligonucleotide comprising aunique identifier sequence for the cellular component-binding reagent,and wherein the cellular component-binding reagent is capable ofspecifically binding to at least one of the plurality of cellularcomponent targets, and wherein the cellular component-binding reagentspecific oligonucleotide comprises one or more of: (a) a secondmolecular label sequence; (b) an alignment sequence adjacent to apoly(dA) region; (c) a linker, wherein the cellular component-bindingreagent specific oligonucleotide is associated with the cellularcomponent-binding reagent through the linker; and (d) the sequence of afirst universal primer, a complementary sequence thereof, a partialsequence thereof, or a combination thereof; extending barcodeshybridized to the cellular component-binding reagent specificoligonucleotides, or products thereof, to produce a plurality of labelednucleic acids, wherein each of the labeled nucleic acid comprises aunique identifier sequence, or a complementary sequence thereof, and afirst molecular label sequence, or a complementary sequence thereof; andobtaining sequence information of the plurality of labeled nucleicacids, a complementary sequence thereof, or a portion thereof todetermine the number of copies of at least one cellular component targetof the plurality of cellular component targets in one or more of theplurality of cells.
 17. The method of claim 16, comprising prior toextending barcodes: partitioning the plurality of cells associated withthe plurality of cellular component-binding reagents to a plurality ofpartitions, wherein a partition of the plurality of partitions comprisesa single cell from the plurality of cells associated with the cellularcomponent-binding reagents; and in the partition comprising the singlecell, contacting a barcoding particle with the cellularcomponent-binding reagent specific oligonucleotide, wherein thebarcoding particle comprises a plurality of barcodes each comprising atarget binding region and a first molecular label sequence, and whereintwo barcodes of the plurality of barcodes comprise different firstmolecular label sequences.
 18. The method of claim 16, wherein thenumber of unique first molecular label sequences and/or the number ofunique second molecular label sequences associated with the uniqueidentifier sequence for the cellular component-binding reagent capableof specifically binding to the at least one cellular component target inthe sequencing data indicates the number of copies of the at least onecellular component target in the one or more of the plurality of cells.19. The method of claim 16, wherein (a) the alignment sequence comprisesa guanine, a cytosine, a thymine, a uracil, or a combination thereof;(b) the alignment sequence comprises a poly(dT) sequence, a poly(dG)sequence, a poly(dC) sequence, a poly(dU) sequence, or a combinationthereof; and/or (c) the alignment sequence is 5′ to the poly(dA) region.20. A plurality of sample indexing compositions, wherein each of theplurality of sample indexing compositions comprises a cellularcomponent-binding reagent associated with a sample indexingoligonucleotide, wherein the sample indexing oligonucleotide comprisesone or more of: (a) a first molecular label sequence, optionally whereinthe first molecular label sequence is 2-20 nucleotides in length; (b) analignment sequence adjacent to a poly(dA) region, optionally wherein thealignment sequence is one or more nucleotides, or two or morenucleotides, in length; and (c) a linker, wherein the sample indexingoligonucleotide is associated with the cellular component-bindingreagent through the linker wherein the cellular component-bindingreagent is capable of specifically binding to at least one cellularcomponent target, wherein the sample indexing oligonucleotide comprisesa sample indexing sequence for identifying sample origin of one or morecells of a sample, and wherein sample indexing sequences of at least twosample indexing compositions of the plurality of sample indexingcompositions comprise different sequences.
 21. A method for sampleidentification, comprising: contacting each of a plurality of sampleswith a sample indexing composition of a plurality of sample indexingcompositions of claim 20, respectively, wherein each of the plurality ofsamples comprises one or more cells each comprising one or more cellularcomponent targets, wherein the sample indexing composition comprises acellular component-binding reagent associated with a sample indexingoligonucleotide, wherein the cellular component-binding reagent iscapable of specifically binding to at least one of the one or morecellular component targets, wherein the sample indexing oligonucleotidecomprises a sample indexing sequence, and wherein sample indexingsequences of at least two sample indexing compositions of the pluralityof sample indexing compositions comprise different sequences; andidentifying sample origin of at least one cell of the one or more cellsbased on the sample indexing sequence of at least one sample indexingoligonucleotide of the plurality of sample indexing compositions.